Can stress cause staph infection

Can stress cause staph infection DEFAULT

What Is Staph Infection? Symptoms, Causes, Diagnosis, Treatment, and Prevention

Staph infections are caused by a type of bacteria called staphylococcus. These germs can live on your skin, in your mouth, or in your nose.

There are more than 30 types of staph bacteria, but the most common is Staphylococcusaureus, according to MedlinePlus. (1)About 30 percent of healthy adults have S.aureus in their nose, and about 20 percent have it on their skin, notes the Merck Manual. (2)

Most of the time, staph doesn’t cause a problem. But if the bacteria spreads deeper into your body, you can develop a serious, or even life-threatening, infection.

Types of Staph Infections

There are two types of staph infections, broadly speaking: skin infections and invasive infections. Skin infections are much more common, and most of the time, less serious, although they can be annoying and painful. But they can develop into invasive infections if they’re left untreated. Invasive staph infections can be life-threatening if they’re not managed properly, notes the Mayo Clinic. (3)

Cellulitis, Impetigo, and Other Skin Infections Caused by Staph

Some examples of skin infections that can be caused by staph include:

Folliculitis In folliculitis, the hair follicles (the small pockets in the skin where strands of hair grow) become inflamed and often infected. Men with curly hair who shave their beard close to the skin often get folliculitis on the face and neck.

Stye A stye is a small, red, very painful bump that grows from the base of an eyelash or under the eyelid. There’s usually visible pus in the center of the bump.

Boils Boils are red, swollen, painful lumps that form under the skin. They usually start as an infected hair follicle that enlarges and fills with pus — bacteria, white blood cells, and dead skin. Boils, also called furuncles, often occur on the face, neck, armpits, buttocks, or inner thighs. A cluster of boils that form a connected area of infection is called a carbuncle.

Skin Abscess A skin abscess is a pocket of pus surrounded by a thick membrane under the skin. Abscesses form when the body tries to protect itself from an infection by walling it off. Cutting a hole in the wall of the abscess with a surgical knife and draining the pus is the only real way to treat an abscess.

CellulitisCellulitis is an infection of the deeper layers of the skin, including the dermis, or second layer of skin, and the subcutaneous tissue, or fat and connective tissue that form the bottom layer of skin. Cellulitis can occur anywhere on the body but most commonly affects the lower legs.

Impetigo This common and highly contagious staph infection begins as small blisters, usually on the face, hands, or feet, that eventually develop a honey-colored crust. Impetigo usually affects young children, but anyone can develop it.

Staphylococcal Scalded Skin Syndrome (SSSS) This condition is caused by toxins produced by a staph infection and results in peeling skin over large areas of the body. The skin might look burned or scalded. It most often affects babies and young children.

Wound Infections These infections usually crop up two or more days after you’ve had a skin injury or surgery. (1,2,3)

Learn More About Cellulitis

Learn More About Impetigo

Invasive Staph Infections

Invasive staph infections happen when bacteria enter the bloodstream. Some types are:

SepsisSepsis is an infection of the bloodstream that leads to a widespread inflammatory response. It’s one of the most dangerous outcomes of staph and can spread throughout your body, affecting the function of internal organs.

Septic Arthritis This infection targets your joints, often the knees, hips, shoulders, finger joints, or toe joints.

Toxic Shock Syndrome This rare but serious condition can happen when staph bacteria get into your bloodstream and produce toxins. Toxic shock syndrome has been linked to the use of high-absorbency tampons, but anyone, including men, children, and women who don’t use tampons, can develop it under the right circumstances.

EndocarditisEndocarditis happens when bacteria affect areas in your heart, most typically the heart valves. If it’s not treated quickly, the infection can damage or destroy your heart valves.

Osteomyelitis This uncommon but serious condition happens when a bone in your body becomes infected.

Pyomyositis Pyomyositis is a rare bacterial infection of the skeletal muscles, which are the muscles you use for movement.

PneumoniaPneumonia is a common infection that causes inflammation and fluid accumulation in the air sacs of your lungs, making it very hard to breathe comfortably.

Food Poisoning Food poisoning connected to staph is caused by eating foods contaminated by toxins that are produced by the bacteria. It is not a true bacterial infection and should not be treated with antibiotics. Foods typically becomes contaminated by being touched by people who have staph on their hands. (1,2,3)

Drug-Resistant Infections: MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) is a type of staph infection that’s resistant to commonly used antibiotics, according to the American Academy of Family Physicians. (4)

Most cases of MRSA occur in people who’ve been in hospitals or other healthcare facilities. But so-called community-associated MRSA, in which the infection is spread outside of hospitals or healthcare facilities, also occurs.

Overuse and misuse of antibiotics are contributors to the development of MRSA. On an individual level, you can help prevent and control the spread of antibiotic resistance by taking antibiotics only when necessary (not for viral infections like the flu or colds) and when they are prescribed to you by a doctor.

Also, always finish your full course of antibiotics, even if your symptoms have improved, to prevent the development of antibiotic-resistant superbugs, such as MRSA.

Learn More About MRSA Symptoms, Diagnosis, and Treatment

Sours: https://www.everydayhealth.com/staph-infection/guide/

A mechanobiological discovery made by using an atomic force microscope creates prospects in the fight against nosocomial diseases caused by staphylococci.

Sometimes referred to as a ‘hospital killer’, golden staphylococcus (Staphylococcus aureus) is, along with another well-known bacterium, Escherichia coli, responsible for the majority of nosocomial diseases: diseases that are contracted in hospital. Staphylococci, which are often found in dirty water, soils, etc., and can also be responsible for food poisoning, are regulars on our skin and external mucosa: according to the Institut Pasteur, 30 to 50% of humans are healthy carriers of staphylococci, meaning they’re carriers of the bacteria without developing disease. The bacteria are therefore considered members of our natural bacterial flora.

Then when do they cause infections? Apart from contaminated food ingestion, which doesn’t concern us here, contaminations can occur during injuries, when the skin is broken, and surgery. The resulting pathologies are, however, most often controlled by antibiotic treatment. But not always. Firstly because many staphylococcus strains have become resistant to this type of treatment, but secondly because staphylococci have the unfortunate tendency to form biofilms, which for years have been the target of Professor Yves Dufrêne, an FNRS research director at UCL’s Louvain Institute of Biomolecular Science and Technology (LIBST) as well as a Welbio researcher.

‘Biofilms are communities of bacteria that can contain several billion of them’, Prof. Dufrêne explains. ‘They adhere to the surfaces of human tissues but also the surfaces of medical devices such as catheters, probes or prostheses before secreting a coating, multiplying, joining together and forming a very sticky layer that’s very difficult to eradicate’.

It’s indeed curious that while individual bacteria are susceptible to antibiotics, as biofilm, they resist. Biofilms are considered responsible for 60 to 80% of nosocomial infections. The fight against them has become a major medical issue. ‘The challenge of our research,’ Prof. Dufrêne says, ‘is to block the adhesion of bacteria rather than using antibiotics. They don’t adhere by chance, they have developed on the surface of key proteins, called adhesins or adhesion proteins, that have a very specific ‘key-lock’ interaction. Disabling these makes the bacteria non-adherent and that’s what we’re trying to achieve.’

Mechanobiology

In , Prof. Dufrêne’s team discovered that a protein located on the surface of staphylococcus, SasG, plays a role in the adhesion of bacteria to each other: in the presence of zinc, SasG behaves like a Velcro band which attaches to the Velcro of nearby bacteria. Last year, his team took a big step forward, identifying a molecule capable of inactivating a major adhesion protein that allows staphylococci to form biofilms. It’s a peptide derived from the neuronal molecule β-neurexin. These two major discoveries were made possible thanks to the development of a new field of biology: mechanobiology.

The field took off around 20 years ago and involves studying how cells react to mechanical forces. It was born out of a question: How do we understand the mode of action of cells in their biochemical environment if we neglect their ability to exert mechanical forces and react to them? This was essential, because cells use these tensions and pressures to adapt to their environment, for example by migrating or otherwise adhering to a material. In fact, the interactions of cells with each other or with their environment are influenced by the mechanical stresses exerted on them. Thus, for example, epithelial cells, such as those that make up skin or intestinal walls, have been observed communicating via mechanical forces to coordinate their movements. Another example: researchers have found that stretching cells increases the activity of two intracellular proteins (YAP and TAZ), causing uncontrollable cell replication, as if they were becoming ‘cancerous’. Still another: cells that have space around them continue to divide, while those that are confined do so much more slowly. It’s understandable that this area of biology increasingly targets areas as varied as oncology, embryo development, scarring, and, more recently, microbiology, and, more particularly, the formation of bacterial biofilms. 

Atomic force microscope 

These advances have been possible thanks to technological developments in live-cell nanoscopy. ‘In our work,’ Prof. Dufrêne explains, ‘as in the work of many others in mechanobiology, we must observe the behaviour of proteins, which are much smaller than cells. On the order of one nanometre, one-millionth of a millimetre, which is not observable by optical microscopes.’

In optical microscopy, magnification is limited by the wavelength of light. Electron microscopy, first introduced in , allows much higher magnifications, using electrons rather than photons. More recently, new microscopies ‘with local probes’ have appeared, allowing for the first time to ‘see’ and ‘feel’ atoms and molecules. The principle is radically different from that of other microscopes because, instead of using incident radiation (photons, electrons), a microscopic probe is moved at a very short distance from the surface to be analysed. Thus, in biology, the atomic force microscope (AFM) allows for the observation of life at the nanoscale as well as directly in a liquid medium, which the electron microscope doesn’t allow. Invented in by the physicists Heinrich Rohrer, Gerd Binning and Calvin Quate, the AFM performs 3D mapping and describes the physical properties of a material at the nanoscale. To achieve this, it detects atomic forces between the surface of the sample and a probe, or tip, that moves at a very short distance above it. The tip, which must be as minute as possible, is at the end of a flexible cantilever that records any disturbances and – especially interesting to us here – measures observed forces.

Stress increases strength tenfold

‘If the bacteria join each other or walls’, Prof. Dufrêne said, ‘it’s because proteins on their surface develop adhesion forces. Our goal was to measure these forces through the AFM to better understand how these proteins work. It is exceptional to achieve this with live bacteria.’ With what results? At rest, the forces observed are obviously very low (on the order of the pico (10N)), but this value is multiplied by several orders of magnitude when the protein is ‘stressed’, for example when it’s stretched. Explains Prof. Dufrêne, ‘We then reach values ​​equivalent to a . Measuring this in living cells is quite exceptional and unexpected.’ In other words, the researchers have observed a major phenomenon: the adhesiveness of adhesion proteins is strongly enhanced when they’re subjected to mechanical stress. Yet this stress is exactly what characterises the daily lives of our staphylococci: whether in the bloodstream, on catheters, or on skin (when we scratch ourselves, for example) or even when they multiply into biofilms, they’re subject to turbulence and shocks.

In their latest study published in the journal PNAS(1) and carried out with colleagues from Trinity College Dublin, the researchers were interested in a particular adhesion protein, ClfA, which has the particularity of functioning in ‘key-lock’ interaction with fibrinogen, a blood plasma protein widespread on medical devices. In the event of mechanical tension or stress, this key-lock mechanism that binds the two types of proteins is considerably strengthened and the staphylococcus adheres more than ever to the biomaterial. The study was funded by Welbio and the European Research Council.

Henri Dupuis

(1)Staphylococcus aureus clumping factor A is a force-sensitive molecular switch that activates bacterial adhesion, Ph. Herman-Bausier et al., PNAS, May 7, https://doi.org//pnas
AFM operation

This microscope does not use incident rays but a probe consisting of a lever ending in the finest point possible, ideally a single atom. If this atom-tip is close to the atoms of the surface of the sample, a repulsive force will be generated which increases rapidly with the inverse of the distance because the electronic clouds of the ions will overlap. If, however, tip and sample are more distant (but ‘aware’ of each other’s presence), an attractive force (called Van der Waals) will bring them closer together. By moving the tip over the sample (‘scanning’ the sample), one thus obtains a succession of attractions and repulsions which is clearly recorded by the lever and generates a 3D map of the surface.

A glance at Yves Dufrêne's bio

Yves Dufrêne

                      Graduated from Faculty of Bioscience Engineering, UCL
                      Doctoral thesis, UCL
                 Post-doctorate at Naval Research Laboratory (US)
Since             FNRS Researcher (Senior Research Associate), Institute of Life Sciences, and Professor in the Faculty of Bioscience Engineering, UCL
                      Winner, Prix Pacault, Société française de Chimie (‘French Society of Chemistry’)
                      Winner, Quadrennial Life Sciences Award, European Microscopy Society
                      Winner, Prix Léo Errera, Royal Academy of Belgium
                      Associate Editor, Nanoscale
                      Winner, European Research Council Advanced Grant for research project NanoStaph

Sours: https://uclouvain.be/en/sciencetoday/news/stressed-out-staph.html
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Khloe Kardashian says stress made her staph infection battle more difficult

Her fans were fearing for her health after she was laid up in hospital after suffering a nasty staph infection.

AndKhloe Kardashian revealed her stressful lifestyle only made her symptoms worse as she gave and update on her condition for her supporters to enjoy  on social media on Saturday.

The year-old further eased fears for her health by insisting she was on the mend, in part due to the 'tons of love' she was receiving from family, friends and followers.

Scroll down for video 

Stress related illness: Kardashian revealed her hectic lifestyle has made her staph infection recovery more difficult; here she is seen on October 12, the day before Lamar OD'd

Stress related illness: Khloe Kardashian revealed her hectic lifestyle has made her staph infection recovery more difficult

The reality television personality tweeted: 'Staph infection is no joke. Mine was exacerbated by stress which is not a great combo. 

'I'm under great medical care and receiving tons of love!

'I appreciate all of your prayers, well wishes and love being sent my way. I'll be better very soon. I love you guys.'

The self-righteous star also pleaded with her army of followers to be nice to one another.

See more of the latest news, pictures and video updates on Khloe Kardashian 

So thankful: She praised her family, friends and followers for all of their love and good wishes

So thankful: She praised her family, friends and followers for all of their love and good wishes

She said: ' Please everyone take care of yourselves and pay attention to your bodies! Be kind and loving to one another! It goes a long way!'

Khloe's family has been 'freaking out' about about her illness, according to TMZ, but she have an indication on Friday she was feeling better already.

The Keeping Up With The Kardashians star announced on her site KhloeWithAK.com she is hosting Thanksgiving this year for her family.

Getting better: On Friday Khloe also announced on her site KhloeWithAK.com she is hosting Thanksgiving

Getting better: On Friday Khloe also announced on her site KhloeWithAK.com she is hosting Thanksgiving

Ready to host: In a post titled The Hostess With The Mostest, she wrote, 'Thanksgiving is at my house this year!!!' The star lives in the same gated community, The Oaks in Calabasas, as sister Kourtney

Ready to host: In a post titled The Hostess With The Mostest, she wrote, 'Thanksgiving is at my house this year!!!' The star lives in the same gated community, The Oaks in Calabasas, as sister Kourtney

In the amusingly titled post The Hostess With The Mostest, she wrote, 'Thanksgiving is at my house this year!!!' The star lives in the same gated community, The Oaks in Calabasas, as sister Kourtney.

'This is the second time I'm doing dinner in my home and I like that it's my tradition,' the Strong Looks Better Naked author wrote.

The blonde beauty added, 'I start preparing the day before and then, on Thanksgiving Day, I start at 7 a.m. It's just fun and I love to cook. (P.S. My famous mac & cheese recipe is going to be on my app next week!!!).'

See the latest news on Khloe Kardashian on her Thanksgiving plans 

She does it right" 'This is the second time I'm doing dinner in my home and I like that it's my tradition,' the Strong Looks Better Naked author wrote

She does it right" 'This is the second time I'm doing dinner in my home and I like that it's my tradition,' the Strong Looks Better Naked author wrote

Yummy: The blonde beauty added, 'I start preparing the day before and then, on Thanksgiving Day, I start at 7 a.m. It's just fun and I love to cook. (P.S. My famous mac & cheese recipe is going to be on my app next week!!!)'

Yummy: The blonde beauty added, 'I start preparing the day before and then, on Thanksgiving Day, I start at 7 a.m. It's just fun and I love to cook. (P.S. My famous mac & cheese recipe is going to be on my app next week!!!)'

Baked goods: And the daughter of Kris Jenner, 60, likes to make it fancy. 'I really like to get my table-setting just right,' she added. 'You won't see porcelain turkeys or anything like that on my table. I want the pictures to be timeless and besides, the food should shine'

Baked goods: And the daughter of Kris Jenner, 60, likes to make it fancy. 'I really like to get my table-setting just right,' she added. 'You won't see porcelain turkeys or anything like that on my table. I want the pictures to be timeless and besides, the food should shine'

Cozy quarters: The knockout in the mini dining room of her mansion, which Justin Bieber once owned

Cozy quarters: The knockout in the mini dining room of her mansion, which Justin Bieber once owned

Inviting: The Dash Dolls guest star often has lit candles and fresh flowers in her massive home

Inviting: The Dash Dolls guest star often has lit candles and fresh flowers in her massive home

And the daughter of Kris Jenner, 60, likes to make it fancy.

'I really like to get my table-setting just right,' she added.

'You won't see porcelain turkeys or anything like that on my table. I want the pictures to be timeless and besides, the food should shine. Instead, I do floral centerpieces that I can re-use after Thanksgiving. Here are some of my fave items that will help you have a very KoKo Thanksgiving!!!

She likes her sweets: Last month the sister of Kim and Kourtney shared a look at her pantry that was full of sugary cereal and candy

She likes her sweets: Last month the sister of Kim and Kourtney shared a look at her pantry that was full of sugary cereal and candy

Getting better: Kourtney Kardashian has revealed her sister Khloe is 'doing ok' as she fights off a staph infection

Getting better: Kourtney Kardashian has revealed her sister Khloe is 'doing ok' as she fights off a staph infection

Kourtney has revealed that Khloe 'doing ok' as she continues to recover from her illness.

Speaking on the red carpet at the  Variety and Women's Wear Daily luncheon, on Thursday she told E! News: 'She's good. She's Ok. She's not feeling well, but she's OK.' 

See the latest Kourtney Kardashian news on Khloe Kardashian and her family

The reality star is being treated with intravenous antibiotics and has cancelled her book tour after picking up the hospital-borne infection.

Khloe's grandfather Robert Houghton died of a staph infection that he picked up after a car crash back in

Busy schedule: Khloe has been doing almost daily in-store appearances to promote Strong Looks Better Naked as well as lots of press. The star is seen here last week at an appearance in San Diego

Busy schedule: Khloe has been doing almost daily in-store appearances to promote Strong Looks Better Naked as well as lots of press. The star is seen here last week at an appearance in San Diego

Kris was sent 'into a panic' and left her 'freaked out', Kardashian sources told TMZ.

Khloe cancelled her book tour on Wednesday to undergo a series of tests after falling ill - and initially blaming her symptoms on stress.

Now it has emerged that the reality star contracted the serious infection during the weeks spent in hospital tending to NBA star Lamar Odom.

She has a lesion on her leg which became painful and she later developed other symptoms including a high fever and swollen glands, TMZ reports. Khloe has been given a course in antibiotics. 

While doctors are optimistic about Khloe's chance of a full recovery, they have banned her heavily pregnant sister Kim from visiting - in case she picks up the infection. 

Khloe took to Twitter on Wednesday to tell fans she was cancelling two public appearances to promote her book Strong Looks Better Naked. 

Making waves: The star's book is now a New York Times Best Seller and details her path to fitness and her recovery from her marriage collapse

Making waves: The star's book is now a New York Times Best Seller and details her path to fitness and her recovery from her marriage collapse

At the time, the star thought stress was to blame, writing: 'Dolls! So sorry I needed 2 re schedule my book signing. I'm sick & dr's orders are that I need to lay low until we narrow down what's wrong.

'I'll be going for more testing today. I'm very sorry again. Stress can run our bodies down so please everyone take care of yourselves.'

She later tweeted: 'I'm reading all of your tweets + messages on my Facebook seriously u guys give me strength! I love and appreciate you more than u know.' 

And she added: 'Signing off Antibiotics +painkillers + tweeting does not equal anything good lol'

The star's representative also confirmed her illness, telling Gossip Cop: 'She is getting treatment and will be OK,' adding she 'just needs to rest and get it treated.' 

The reality star is hoping to reschedule her appearances. 

Staph infections are caused by a bacteria that typically lives on the nose or skin but can turn deadly if it enters deeper into the body and infects the bloodstream, joints, bones, lungs or heart.

Symptoms can vary from painful skin rashes and boils to dangerous heart infections. Food poisoning, blood poisoning and toxic shock syndrome are some of the most common types of staph infections, which are typically treated with antibiotics.

Khloe was meant to be in Houston, Texas, on Friday for a book signing.

The reality star has been doing almost daily in-store appearances to promote her now New York Times Best Seller as well as lots of press.

The Keeping Up With The Kardashians star has also been at estranged husband Odom's bedside almost every day since he collapsed and slipped into a subsequent coma last month.

Tough time: The Keeping Up With The Kardashians star has also been by estranged husband's bedside every day since he collapsed and slipped into a subsequent coma last month (pictured )

Tough time: The Keeping Up With The Kardashians star has also been by estranged husband Lamar Odom's bedside every day since he collapsed and slipped into a subsequent coma last month (pictured )

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Sours: https://www.dailymail.co.uk/tvshowbiz/article/Khloe-Kardashian-says-stress-staph-infection-battle-difficult-healing-thanks-tons-love.html
UNMC- Durham Research Center II -Staph infections research

In today's unpredictable world, stress and chaos are bound to arise. When stress raises its unappealing head, both emotional and physical changes can occur. Of those uncomfortable changes, boils (skin abscesses), can appear and be very annoying.

About Stress-Related Boils

A boil, medically known as a furuncle, is an infection that begins in an enclosed, superficial bodily orifice, such as an oil gland or hair follicle. It begins as a tender, inflamed area. After about a week, it becomes a pus-filled lump or bump. WebMD states the most common area for boils to occur is the face, neck, armpits, shoulders, and buttocks. When many form together, it is referred to as a carbuncle.

Related Articles

Boils occur when bacteria becomes trapped in a follicle or gland. The body naturally has bacteria on the outer surface, but healthy skin keeps bacteria separated from the internal environment of the body. The bacteria can enter the body through a disturbance in the skin, such as a scratch, and continues to replicate if the immune system is unable to counteract the effects immediately.

Emotions like stress can potentially suppress the immune system according to a meta-analysis of 30 years of data. Stress induces hormonal changes in the body, causing the skin to be more sensitive and reactive. According to Harvard Health, boils can be especially noted in immunocompromised populations and are commonly caused by staph aureus, which is found naturally on the skin. Likewise, those with recurrent ingrown hairs are prone to stress boils. If the infection is very prominent, WebMD conveys infection and swollen lymph nodes can ensue.

Treatment Options

Once a stress boil appears, home treatment options are simple enough unless the bacterial infection is highly elevated. Stress boils usually resolve on their own with simple intervention, and the prognosis is good. However, when starting any new medical treatment regimen, always check with a medical expert before taking over-the-counter medications.

Relieve Stress

Calm down. Alleviate factors that are causing stressful situations in day-to-day activities. Incorporate stress management activities like deep breathing exercises or meditation that integrate components of peace and pleasantry into life.

Use Warm Compresses

Dr. Melissa Stoppler, MD suggests warm compresses or soaks several times a day to increase circulation and bring healing cells to the area of the boil. Do not poke or press with a sharp object for risk of further transferring more bacteria.

Drain It

Once the boil comes to a head, it is usually ready to drain. Smaller boils usually drain on their own from the warm compresses, and pain relief ensues from the release of the pressure. For larger boils, medical professional lancing/draining maybe indicated as again noted by Dr. Stoppler. The larger boils may contain many areas of pus that need to be lanced.

Use Antibiotics

When the stress boil is accompanied by additional surrounding skin infection and a temperature, a physician may prescribe antibiotics. Antibiotics work in conjunction with the lancing of the lesion. A topical antibiotic ointment may also be prescribed to address the superficial layers of skin. Boils located on the face, spine, groin, or in the rectum may also require medical attention.

Pain Management

Usually, the pain accompanying the stress boils and the surrounding areas come from the pressure build up of the pus underneath. With smaller boils, the warm compresses usually lead to drainage, which relieves pressure. The professional lancing of larger boils also relieves pain from pressure. Ultimately, until the necessary lancing takes place, pressure from the pus build-up can be addressed with the usual preferred over-the- counter drug of choice, such as anti-inflammatories or a numbing topical.

Avoiding Stress Boils

In both the regular population and in immunocompromised patients, one can take basic steps to reduce stress boils. Keep bacteria under control through hand and skin washing, keeping cuts covered, and not exchanging personal items for use, such as razors and towels, notes Mayo Clinic. Reducing stress is also important, as is proper diagnosis and treatment of underlying immunosuppressive conditions, such as diabetes and cancer.

Keep Calm and Stay Clean

Though furuncles are common, stress-induced boils and the underlying causes can be particularly annoying and uncomfortable. Reducing daily stressors, keeping clean healthy skin, and overall maintaining an immune system that is healthy as possible will reduce the chances of reoccurrence of this nuisance condition.

© LoveToKnow Media. All rights reserved.

Sours: https://stress.lovetoknow.com/about-stress/dealing-stress-related-boils

Staph cause can infection stress

Learn About Stress-Induced Infectious Diseases

Severe, prolonged chronic stress has negative effects on the human body, including increasing the risk of becoming sick from infections.

Can Stress Really Increase Your Risk for Infections?

Studies have shown that people with greater levels of continuous stress are more prone to getting some infectious diseases.

It is important to note, however, that stress levels differ from person-to-person, due to individual differences in a person’s emotional and physiological makeup. Therefore, a situation that causes significant stress for one person may or may not have the same effect on another.

What Happens to Your Body When You Are Stressed?

  • Acute stress response: The acute stress response is an immediate response to a stressful event. The body’s immediate response is to release stress hormones, which help the body to make energy. This energy is diverted to muscle and brain tissues, and certain cells of the immune system may become more active.
  • Chronic stress response: Chronic stress occurs when a person has continuous acute stress responses. Chronic stress results in more sustained changes in the body, such as increased blood pressure, which over time can result in damaged arteries and heart disease. The continuous increase in stress hormones might also result in suppression of the immune system’s white blood cells, leading to an increased risk of infections.

Your Immune System's Reaction to Infections

Your body has an innate immune response, which is the first line of defense that provides an immediate and non-specific response to infectious pathogens (such as bacteria and viruses). Shortly afterwards, your body begins to generate its adaptive immune response, in which pathogens are specifically targeted and attacked by white blood cells.

  • Acute stress: Researchers have found that during periods of acute stress, cells of the innate immune system are more active and increase their circulation throughout the body in order to patrol for infectious microbes.
  • Chronic stress: During periods of chronic stress, the adaptive immune system is suppressed by high levels of stress hormones. As a result, your body might heal slower, could be less able to produce antibodies, and may be more susceptible to illness from infections.

Which Infections Are You More Likely to Get?

Studies have linked chronic stress with tuberculosis, herpes simplex virus reactivation, shingles, ulcers (caused by infectious Helicobacter pylori bacteria), and other infectious diseases.

Some studies of vaccinations have shown a decrease in effectiveness in individuals with high chronic stress. However, the size of the effect that stress really has on infection risk is not clear.

Common Cold: A classic study at Carnegie Mellon University, published in , showed that the risk for the common cold was proportional to the degree of stress in a person’s life.

But stress due to a single, recent event can have a different effect than chronic stress. A subsequent study in showed that people who had chronic stress (due to life events, such as unemployment or interpersonal difficulties) for at least one month were more likely to get the common cold than those who had shorter durations of stress.

AIDS: The HIV virus leads to AIDS, a condition characterized by severe immunodeficiency. Stress doesn't lead to HIV infection, but stress can cause people who have been infected by the HIV virus to experience a quicker progression to AIDS. 

A UNC-Chapel Hill study published in found that men with HIV progressed to AIDS faster if they had chronic stress in their lives. For each increased stressful event, the risk for AIDS progression doubled.

Reducing Your Risk of Infections

See your healthcare provider if you need help in coping with stress. There are numerous strategies recommended for coping with stress, including psychosocial interventions that decrease a person’s own perception of stress and improves their social supports. Certain medications may also help in reducing the stress that's caused by specific disorders.

How each person responds to stress varies. Some people deal with stress by engaging in unhealthy health behaviors, such as smoking, drinking, or excessive eating — all of which will contribute to the chances of getting infections. And in some cases, these health behaviors contribute to worse stress, resulting in a continuous cycle of poor health and stress. It is important to remember, though, that there are many factors involved in getting an infectious disease, and stress management should be accompanied by healthy habits, such as hand washing and avoiding exposure to infections.

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Sours: https://www.verywellhealth.com/stress-induced-infectious-diseases
Vital Signs: Staph infections can kill

Stress-induced inactivation of the Staphylococcus aureus purine biosynthesis repressor leads to hypervirulence

Abstract

Staphylococcus aureus is a significant cause of human infection. Here, we demonstrate that mutations in the transcriptional repressor of purine biosynthesis, purR, enhance the pathogenic potential of S. aureus. Indeed, systemic infection with purR mutants causes accelerated mortality in mice, which is due to aberrant up-regulation of fibronectin binding proteins (FnBPs). Remarkably, purR mutations can arise upon exposure of S. aureus to stress, such as an intact immune system. In humans, naturally occurring anti-FnBP antibodies exist that, while not protective against recurrent S. aureus infection, ostensibly protect against hypervirulent S. aureus infections. Vaccination studies support this notion, where anti-Fnb antibodies in mice protect against purR hypervirulence. These findings provide a novel link between purine metabolism and virulence in S. aureus.

Introduction

In humans, Staphylococcus aureus may exist as a commensal bacterium or as a pathogen. Data from the United States Centers for Disease Control and Prevention show that approximately one-third of the US population is colonized with S. aureus1, and colonization with S. aureus is associated with increased risk of subsequent infection2. Infections caused by S. aureus range in severity from relatively minor skin and soft tissue infections through to invasive diseases such as pneumonia, infective endocarditis and osteomyelitis3. Strikingly, the magnitude of morbidity and mortality caused by S. aureus is highlighted by reports that, in the US, invasive infections by this bacterium cause more deaths than HIV4.

That S. aureus can infect virtually any organ or tissue in the body is a reflection of its vast repertoire of virulence factors that contribute to bacterial pathogenesis through mechanisms involving tissue adherence5,6, cellular intoxication7,8,9, and immune modulation and deception10,11. Virulence factor expression in S. aureus is complex and coordinately regulated by multiple transcription factors, regulatory RNAs, two-component sensing systems and quorum-sensing12,13,14. Despite a wealth of knowledge on virulence regulation in S. aureus, there are still outstanding questions to be resolved, as novel mechanisms of virulence regulation are still being discovered, especially with regard to environmental or metabolic cues to which S. aureus responds15.

Exposure to elevated temperatures, for example 42 °C, a temperature frequently used to cure S. aureus of recombinant plasmids during mutagenesis procedures, can select for mutations in the S. aureus genome. Mutations in the global two-component regulator SaeRS have previously been isolated following mutagenesis16, and mutations in the sae regulatory system show drastically reduced toxin production and have attenuated virulence17,18,19,20. Screening for unintended sae mutations is straight forward, as the mutants are easily identified as having reduced haemolytic activity on blood agar plates. Little is known, however, about other unintended secondary mutations that may be selected for in response to stress, especially those that may impact on the virulence potential of S. aureus.

In this study, we identify mutations that occur in the S. aureus purR gene in response to stress, including growth at elevated temperatures (i.e. 42 °C). The function of purR in S. aureus has not been characterized, but the gene is homologous to those that encode the purine biosynthesis repressors in Bacillus subtilis and Escherichia coli; we show here that mutations in purR do indeed result in upregulation of purine biosynthetic genes in S. aureus. However, we further demonstrate that purR mutations have a heretofore undescribed role in regulating expression of S. aureus fibronectin binding proteins and, thus, play a role in the interaction of S. aureus with fibronectin. In the absence of anti-FnbA/B antibodies, purR mutants clump vigorously in serum in a fibronectin-dependent manner. Importantly, purR mutants, also through FnbA/B-dependent mechanisms, are hypervirulent in a systemic model of infection in mice, and vaccination of mice against FnbA/B can diminish hypervirulence and ameliorate animal mortality. We suggest that, in S. aureus, PurR is critical to limit the expression of fnb genes, known to be maximally expressed at low cell density, in order to promote colonization, yet prevent cell clumping in the vasculature.

Results

S. aureus purR mutants vigorously clump during growth in serum

In our laboratory, we generate deletion mutations in iron-regulated genes and test mutants for growth in chemically defined media (e.g. RPMI) containing 10% v/v horse serum (HS) to induce iron starvation. Over time, we noted that a number of mutants, in the USA genetic background, would clump vigorously when grown in the presence of HS, a trait not observed for WT USA The hallmark of this phenotype was that, during growth, visibly large clumps would appear in the culture and, when the culture tube was allowed to sit without shaking, the clumped material would settle to the bottom of the tube within minutes. This response was independent of iron starvation as robust clumping occurred when the bacteria were grown in tryptic soy broth, an iron replete medium, containing 10% v/v HS (TSB-S). To investigate this phenotype further, we performed whole genome sequencing on one of these clumping mutants and identified a non-synonymous single nucleotide polymorphism (SNP) in the purR gene causing a Q52P mutation (purRQ52P). The purR gene is homologous to those encoding the purine biosynthesis repressors in E. coli and B. subtilis but, to date, has not been studied in S. aureus. We independently discovered a second clumping mutant while generating a completely separate markerless deletion in the USA genome. We PCR-amplified the purR gene and discovered it carried a deletion of a guanine at position of the gene, causing a frameshift in the protein after V To confirm that loss of purR indeed correlated with the hyper-clumping phenotype, we mobilized the purR::ΦNΣ mutation from the Nebraska transposon mutant library21 into our laboratory USA strain (hereafter referred to as purR::ΦNΣ). The purR::ΦNΣ strain demonstrated similar clumping to the SNP-containing strain and the phenotype could be fully complemented by providing purR in trans on a multi-copy plasmid (referred to as ppurR) (Fig. 1a). Given that cultures containing clumped bacteria, when allowed to sit without shaking, rapidly clarify due to sedimentation of the cells in culture tubes, we developed an assay to quantitate relative clumping by measuring the culture optical density (see the Methods section). This analysis detected a significant decrease in OD values for both WT USA and purR::ΦNΣ in TSB-S, when compared to TSB alone. However, bacterial sedimentation (i.e. clumping) was greatly enhanced for purR::ΦNΣ in serum as compared to WT USA (Fig. 1b). Furthermore, these measurements confirmed that provision of purR in trans completely reversed the clumping phenotype (Fig. 1b).

Disruption of purR causes cell clumping of S. aureus USA In a, representative images of USA, USA purR::ΦNΣ or the complemented purR::ΦNΣ mutant in culture tubes following growth in TSB with 10% (v/v) horse serum (TSB-S) for  h from a starting OD equivalent of In b, graphical representation of the relative sedimentation of bacterial aggregates in cultures as grown in a, reflected by the OD values of the centre of liquid cultures after sitting without shaking for 5 min following shaking at 37 °C for  h. Data are mean ± SEM of four independent experiments. ***p-value < , based on a one-way analysis of variance (ANOVA) with a Bonferroni post-test. In c, the representative micrographs show bacterial cell clusters that arise during growth in TSB or TSB-S. White boxes define the region of interest that is depicted in the insets. Scale bars  = 40 µm. In d, transmission electron micrographs are shown for S. aureus USA and the USA purR::ΦNΣ strain grown in the presence (TSB-S) or absence (TSB) of horse serum. The representative images depict cells at ×11, magnification. Scale bars  = 1 µm. Source data are provided as a Source Data file

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To study the hyper-clumping phenotype further, we used brightfield microscopy to examine the cells grown in TSB or TSB-S. WT USA and the complemented purR::ΦNΣ mutant grown in TSB formed only small ‘grape-like’ clusters of 2–4 cells, as expected for S. aureus. In contrast, purR::ΦNΣ formed aggregates comprised of greater numbers of cells, including some noticeably larger clusters that were not observed for WT bacteria (Fig. 1c, top panels). Consistent with what is known of the interaction of S. aureus with serum proteins22,23, USA and the purR::ΦNΣ complemented strains, grown in TSB-S as compared to TSB alone, formed larger cell clusters due to aggregation of the bacteria through binding of serum proteins (Fig. 1c, bottom panels). In contrast, bacterial aggregation was greatly exaggerated for purR::ΦNΣ, where aggregated masses of bacteria took up the majority of the field of view (Fig. 1c, bottom panel), undoubtedly related to the macroscopic sedimentation observed in liquid cultures.

To assess whether cell clumping could be caused by cell division defects in the purR::ΦNΣ background, we performed transmission electron microscopy of WT or purR::ΦNΣ mutant cells grown in TSB or TSB-S. For both strains, irrespective of culture conditions, division septa were visible and the apparent cell morphology did not differ, indicating cell division defects were not present in purR::ΦNΣ (Fig. 1d). We therefore next hypothesized that the robust aggregation of purR bacteria was mediated by specific bacterial factors. Interestingly, no discernible differences in protein profiles or growth were observed between WT and purR::ΦNΣ bacteria at various growth phases (Supplementary Figure 1a, b, c). RNAseq on mid-exponential (OD = ) phase cultures grown in TSB showed that genes of the purine biosynthesis pathway were elevated in the purR::ΦNΣ strain, as compared to the WT, however, few other differences could be detected between the two genotypes (Supplementary Table 1). RT-PCR was performed on a select number of genes and the data agreed with the RNAseq data, where purE, the first gene in the purEKCSQLFMNHD purine biosynthetic operon, demonstrated the greatest transcriptional increase (Supplementary Figure 1d). Altogether, these data demonstrate that purR regulates the purine biosynthesis pathway of S. aureus and inactivation of purR leads to exaggerated serum-dependent cell clustering. However, these analyses failed to identify an obvious effector responsible for the clumping phenotype.

Serum clumping requires fibronectin binding proteins

We next analysed whether the purR phenotype was conserved across different S. aureus backgrounds. To this end, we transduced the purR::ΦNΣ mutation into S. aureus strains RN, SH, MN8 and Newman and complemented each mutant. Similar to USA, growth of the RN, SH and MN8 purR mutants in TSB-S demonstrated vigorous cell clumping and, for each strain, provision of purR in trans complemented the phenotype (Supplementary Figure 2a, b, c). In contrast, Newman purR::ΦNΣ failed to hyper-aggregate in the presence of HS and was indistinguishable from WT Newman when grown in either TSB or TSB-S (Supplementary Figure 2d). Of note, strain Newman expresses mutated fibronectin binding proteins (FnBPs; FnbA and FnbB) that, unlike in other S. aureus strains, are not cell wall anchored24, leading us to hypothesize that cell wall anchored FnbA/B may be required for hyper-clumping.

To directly test the involvement of the FnBPs in purR-dependent clumping, we engineered, in WT and purR::ΦNΣ USA bacteria, markerless deletions of the tandemly-duplicated fnbA and fnbB genes. Growth of the resulting purR::ΦNΣ fnbA/B mutants in TSB and TSB-S did not differ from that of WT USA and, notably, serum-dependent hyper-clumping did not occur (Fig. 2a, b). Indeed, the USAΔfnbAB construct exhibited less serum-dependent clumping than WT, demonstrating the importance of these proteins in normal interactions of S. aureus with serum components (Fig. 2a). Of note, complementation of the ΔfnbAB mutants with fnbA on an overexpression plasmid resulted in exaggerated clumping during growth in TSB without serum (Fig. 2a, b), likely due to the increased number of homophilic interactions between FnbA molecules, which have previously been reported to contribute to bacterial aggregation25. Overall, these data show that hyper-clumping due to purR inactivation occurs in several S. aureus strains and requires cell wall-anchoring of FnBPs.

The purR-dependent clumping phenotype requires fibronectin binding proteins and host fibronectin. In a, cultures were grown in TSB or TSB-S for  h and then imaged on a wide field microscope at ×40 magnification. White boxes define the region of interest that is depicted in the insets. Scale bars = 40 µm. Representative images are shown. In b, cultures were grown as in a and OD was measured as described in the legend to Fig. 1 and in the Methods section. Data shown are mean ± SEM of four independent experiments. ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. In c, WT and the purR::ΦNΣ mutant were grown in TSB, TSB-S, TSB containing 10% v/v of various levels of Fn-depleted horse serum or Fn-depleted horse serum with the addition of eluted fibronectin (Fn depletion 3+Fn). Measurement of OD of cultures to evaluate clumping was performed as described above. Data shown are mean ± SEM of five independent experiments and two different Fn purifications. ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. In d, biofilm forming ability of indicated strains was measured after growth in TSB in a standard well plate biofilm assay (see the Methods section). Data shown are mean ± SEM of four experiments. **p-value < , ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. Source data are provided as a Source Data file

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Serum clumping by purR mutants requires fibronectin

The multifunctional S. aureus FnBPs bind to fibrinogen, fibronectin and elastin5. To determine which serum component was involved in the clumping phenotype, we allowed purR::ΦNΣ bacteria to grow in TSB-S and form clumps. We isolated the clumped material and used mass spectrometry to identify enriched serum proteins that copurified with the bacteria (see the Methods section). These analyses revealed only one protein, fibronectin (Fn) from Equus ferus przewalskii (Mongolian wild horse) was significantly enriched in purR::ΦNΣ derived samples. To confirm the involvement of Fn in purR-dependent hyper-clumping, soluble Fn was removed from horse serum by serial passage over a gelatin sepharose column (Supplementary Figure 3). When the Fn-depleted serum was used in clumping assays, we observed a decrease in the hyper-clumping phenotype of the purR::ΦNΣ mutant (Fig. 2c). Reconstitution of the Fn-depleted serum with the purified horse Fn restored purR-dependent clumping to normal levels (Fig. 2c). Together these data demonstrate the purR-dependent clumping in serum requires S. aureus FnBPs and host Fn.

S. aureus purR mutants demonstrate enhanced biofilm formation

The clustering of purR::ΦNΣ cells in TSB, coupled with the dependency of the aggregation phenotype on FnBPs, lead us to hypothesise that purR::ΦNΣ bacteria were better able to initiate biofilm formation. In agreement, purR::ΦNΣ bacteria formed increased biofilm as compared to WT USA (Fig. 2d), and this phenotype could be eliminated by the deletion of fnbAB in the purR::ΦNΣ background (Fig. 2d). Moreover, deletion of fnbAB genes eliminated any differences between WT and purR::ΦNΣ cells and diminished biofilm formation. Conversely, overexpression of fnbA from a plasmid enhanced biofilm formation, irrespective of purR.

PurR represses transcription of the purE operon and fnbAB

How inactivation of purR is connected to FnBP function and/or expression was not understood, since our RNAseq analysis failed to detect changes in either fnbA or fnbB transcript levels at culture densities of OD of Studies in B. subtilis and Lactococcus lactis have identified conserved sequence motifs in promoter regions, named PurBoxes, where PurR binds. Single or double PurBoxes can be present, and double PurBoxes are often palindromic, but all contain a central conserved CGAA motif26,27 (Fig. 3a). Analysis of the S. aureus genome identified sequences similar to those in B. subtilis and L. lactis upstream of the purE and purA genes in S. aureus USA (Fig. 3b) and, not surprisingly, these genes are upregulated in the purR::ΦNΣ strain (see Supplementary Table 1). Remarkably, a similar putative PurR-binding sequence was also present upstream the fnbA and fnbB genes (Fig. 3b). To determine whether transcription of fnbA and fnbB is influenced by PurR we generated plasmids carrying the fnbA and fnbB promoters fused to a promoterless lux-gene construct and monitored bioluminescence in WT and purR::ΦNΣ bacteria. Bioluminescence could not be detected above background levels in WT cells, presumably due to low levels of transcription from the fnbA/B promoters (Fig. 3c). In contrast, bioluminescence was detected for both the fnbA and fnbB promoter constructs in the purR::ΦNΣ mutant, where luminescence peaked at a culture density of OD – (Fig. 3d). Accordingly, we investigated transcript levels of fnbA and fnbB at early growth phases. Relative to WT, fnbA transcripts were elevated in the purR::ΦNΣ mutant, at culture densities as low as OD of (Fig. 3e), and steadily decreased as the culture density increased. Consistent with our RNAseq analysis, no significant differences in fnbA transcripts were present between the WT and the purR::ΦNΣ mutant at an OD of Of note, fnbB transcripts were only elevated in the purR::ΦNΣ mutant at OD of (Fig. 3f). Consistent with de-repression due to the absence of its regulator/repressor, and concordant with our previous data, purE transcripts were upregulated at all time points tested (Fig. 3g).

purR mutations lead to transcriptional upregulation of the purine biosynthesis operon and fnbAB. a Consensus PurBox sequence for B. subtilis and L. lactis (data adapted from refs. 26,27). b Promoter sequences of purE, purA and fnbA, with putative PurBox sequences underlined. WT (c) or purR::ΦNΣ mutant (d) containing a luciferase construct with the promoter sequence of fnbA or fnbB (see the Methods section) were grown in TSB and OD and luminescence monitored. Data shown are mean ± SEM of three experiments. In e, f and g, indicated strains were grown to OD of , or , total RNA was extracted and RT-PCR analysis performed for relative abundance of fnbA (e), fnbB (f) and purE (g) transcripts. All data were normalized to levels of rpoB and expressed as fold change using WT pALC (empty plasmid) as comparator at each OD value. Data shown are mean ± SEM of four independent experiments. *p-value < , **p-value < , ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. Source data are provided as a Source Data file

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S. aureus purR mutants are hypervirulent

Given the strong Fn-binding phenotype associated with a S. aureus purR mutant, we evaluated the virulence potential of the mutant. Mice were infected via the tail vein with WT USA, the purR::ΦNΣ mutant, and the purR::ΦNΣ mutant complemented with ppurR at a dose of ~1 × 107 CFU. Remarkably, % of the mice infected with the purR::ΦNΣ mutant met humane endpoint criteria by 24 hpi, whereas % of the mice infected with either the WT or complemented mutant survived past 72 hpi (Fig. 4a). The purRQ52P mutant demonstrated the same hypervirulent phenotype as the purR::ΦNΣ mutant (Supplementary Figure 4a).

A S. aureus purR mutant is hypervirulent via FnbAB. In a, mice (9–12 per group) were infected with ~1 × 107 CFU of WT USA, USA purR::ΦNΣ or complemented purR::ΦNΣ mutant and survival monitored over 72 h. ***p-value < , based on a Mantel-Cox test. In b, animals were infected as in a, but with 2– × 106 CFU, and c weight loss monitored daily for 48 h. **p-value < , ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. In d, animals from b were sacrificed at 48 h post infection (hpi), and heart, kidney and liver were harvested and bacterial burdens determined. Data shown are mean ± SEM, *p-value < , **p-value < , ***p-value < , based on a Student’s unpaired t-test. In e, two animals per bacterial strain were infected as in a, with ~1 × 107 CFU, sacrificed at 24 hpi and organs harvested. Organs were paraffin embedded, sectioned and stained with H&E and a Gram stain. Representative images are shown. In f, animals were infected as in a, with ~1 × 107 CFU, with the inclusion of WTΔfnbAB and purR::ΦNΣΔfnbAB strains, and monitored for 72 h. ***p-value < , based on a Mantel-Cox test. In g, the heart, kidney and liver from the animals infected in e were harvested at the point of sacrifice and bacterial burden determined. Data shown are mean ± SEM, *p-value < , **p-value < , ***p-value < , based on a Student’s unpaired t-test. Source data are provided as a Source Data file

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In subsequent experiments, we tested the effect of a lower dose of the purR::ΦNΣ mutant and found that infection with ~2 × 106 CFU allowed survival up to 48 hpi (Fig. 4b). At 48 hpi, we observed significantly greater weight loss and increased bacterial burdens in mice infected with the purR::ΦNΣ mutant when compared to those infected with WT (Fig. 4c, d). The mice infected with the complemented strain showed statistically significant decreases in weight loss and bacterial burden, even compared to mice infected with WT.

Histopathological analysis of animals infected with a high dose of WT (~1 × 107 CFU) for 24 h demonstrated lesions predominantly in the heart and the kidney (Fig. 4e). Animals infected with the purR::ΦNΣ mutant had larger and more frequent lesions in both the heart and kidneys (Supplementary Table 2), with multifocal necrotic areas, often centred on discrete groups of Gram-positive bacteria (Fig. 4e). Complementation of the purR::ΦNΣ mutant almost completely eliminated the formation of lesions (Fig. 4e), concurring with the decreased bacterial burden previously observed.

To confirm the role of fnbAB in the purR hypervirulence phenotype, we infected mice with the ΔfnbAB mutant, in either the WT or purR::ΦNΣ background. While purR::ΦNΣ infected animals required sacrifice by 24 hpi the deletion of fnbAB in that background completely ablated the hypervirulent phenotype (Fig. 4f). Of note, infections with strains carrying an fnbA overexpression plasmid, indifferent of the purR background, resulted in very rapid effects on animal health, and animals required euthanasia by ~6–8 hpi (Fig. 4f). This demonstrates the profound effects of aberrant fnb overexpression, suggesting that even transient upregulation of FnBPs has a severe impact on disease severity in a systemic infection model. Bacterial burdens in the hearts, kidneys and livers of the remaining groups were in agreement with the survival data, with increased numbers of bacteria for the purR::ΦNΣ strain, but not for the purR::ΦNΣ ΔfnbAB strain, compared to WT (Fig. 4g) (CFU for pfnbA carrying strains were not determined). Of note, no difference in survival or bacterial burden was observed between the WT and ΔfnbAB strains, indicating that while these proteins are not required for pathogenesis of WT USA in a systemic model of infection, they are indispensable for the hypervirulent phenotype of the purR::ΦNΣ mutant. In agreement with the requirement of FnbAB for purR-dependent hypervirulence, a purR::ΦNΣ mutant in strain Newman was not hypervirulent in this model (Supplementary Figure 4b).

Mutations in purR occur at elevated temperatures and in vivo

The purRQ52P SNP and the purRVframeshift SNP-containing strains were isolated following allelic replacement mutagenesis techniques, a process that has previously been reported to select for mutations in the virulence regulatory genes saeRS16. Plasmids for allelic replacement are often temperature sensitive and curing of plasmids following homologous recombination necessitates growth at elevated temperatures. To investigate if exposure to high temperatures also selects for purR mutations in S. aureus, we constructed a reporter strain that colorimetrically identifies purR mutants. Given that the promoter of the purine biosynthetic operon (purEKCSQLFMNHD) was highly expressed in a purR mutant, we fused the promoter of this operon to a promoterless gusA gene, encoding ß-glucuronidase (referred to as PpurE::gusA) (Supplementary Figure 5a), and inserted this fusion into the genome (see the Methods section). When cultured on X-Gluc-containing solid media, USA PpurE::gusA colonies were pale yellow while the USA purR::ΦNΣ strain carrying the genomic reporter were dark blue; this indicates that the reporter was capable of identifying purR mutants in culture. To define whether we could identify naturally-occurring purR mutants, we cultured USA PpurE::gusA at either 37 °C or 42 °C, with daily passage, for 5 days. Blue colonies were only detected from cultures grown at 42 °C (Supplementary Figure 5b), at a frequency of ~–% following 5 days of passaging (Supplementary Figure 5b). Sequencing of the purR gene from select blue colonies identified a variety of additional mutations in purR (Y71Stop, VE, SStop, HD, SStop, QStop).

The in vivo environment also presents a strong selection pressure on bacteria. Therefore, we were interested to determine if passage of S. aureus through mice would select for purR mutants. Unfortunately, for unknown reasons, the USA PpurE::gusA construct was lost from the genome without antibiotic selection in vivo. Therefore, we tested colonies recovered from the organs of mice infected with WT USA for 4 days for clumping in TSB-S (in a well plate format). Potential mutants were phenotypically confirmed in a tube assay and the purR gene sequenced. A mutant with a R96A SNP was identified from an infected kidney, and demonstrated cell clustering in TSB (Supplementary Figure 5c) and clumping in TSB-S (Supplementary Figure 5d). The phenotype could be complemented by the introduction of ppurR, indicating the SNP was solely responsible for the observed phenotype (Supplementary Figure 5c, d). It was of interest whether strains containing SNPs in purR, and recovered from murine infections, also displayed the characteristic hypervirulence we described here. Infection of mice with the purRR96A SNP resulted in the same hypervirulent phenotype as the purR::ΦNΣ mutant (Supplementary Figure 5e), with animals requiring sacrifice within 24 hpi.

Anti-FnBP antibodies ameliorate purR mutant clumping

To date, despite the hypervirulence phenotype of purR mutation described above, no associations of purR mutations with human infection have been noted. A search of publicly available whole genome sequences identified a non-synonymous change or changes in the purR gene in of sequences (Supplementary Data 1). However, few details on the infection type or outcome were available and, at this point, no correlations could be drawn between the presence of a purR mutation and disease severity. To begin to explore this further in the laboratory setting, we tested whether human serum can support hyper-clumping of purR::ΦNΣ bacteria. We isolated fresh human serum from healthy volunteers and this serum was used to assay for clumping as described above. When the WT and purR::ΦNΣ mutant were grown in TSB with 10% v/v human serum (TSB-HuS), the purR::ΦNΣ clumped but clumping was less pronounced than that seen in horse serum (Fig. 5a). Since the clumping phenotype relies on FnbA and FnbB, and their interaction with Fn, we hypothesised that anti-FnBP (i.e. blocking) antibodies are present in human serum, since humans are exposed to S. aureus throughout their lifetime, and that these antibodies would interfere with clumping. To test this, we passed human serum through a protein A column to remove IgG. TSB containing 10% v/v IgG-depleted human serum showed increased levels of clumping for the purR::ΦNΣ mutant, when compared to TSB-HuS, but no significant difference was observed for the WT (Fig. 5b, c). Moreover, addition of the purified human IgG to the purR::ΦNΣ mutant growing in TSB-S resulted in significant reduction of the clumping phenotype (Supplementary Figure 6). This indicates that antibodies present in human serum can interfere with the purR-dependent clumping phenotype. Western blot analysis determined that human serum did indeed contain anti-FnbA/B antibodies (Fig. 5d); while no signal could be detected for the WT, likely due to low expression, which is in agreement with our luminescence findings (Fig. 3c), bands could be visualised if a strain carried an fnbA overexpression construct. These data indicate that humans carry anti-FnbA/B antibodies that, while not necessarily protective against S. aureus infection, may confer protection against the hyper-virulent purR phenotype.

Anti-staphylococcal antibodies ameliorate purR hyper-clumping. a WT or the purR::ΦNΣ mutant were grown in TSB, TSB-S or TSB with 10% v/v fresh human serum (TSB-HuS) for 3 h and relative clumping ability was measured using OD as described above. Data shown are mean ± SEM of four independent experiments. *p-value < , **p-value <  and ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. WT (b) or the purR::ΦNΣ mutant (c) were grown in TSB (white bars), TSB-S (white bars), TSB-HuS (grey bars) or TSB with IgG-depleted human serum (HuS) (black bars) for 3 h and relative clumping ability measured as above. Data shown are mean ± SEM of four experiments, with four donors. **p-value < , ***p-value < , based on a one-way ANOVA with a Bonferroni post-test. In d, whole cell lysates of WT, WT pfnbA or WTΔfnbAB were used for western blots, with human serum (from donors in panels b and c) or a rabbit anti-Fnb serum (far right blot) used as a source of primary antibody. Source data are provided as a Source Data file

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Anti-FnBP antibodies protect against purR hypervirulence

Given our data indicated that anti-FnbA/B antibodies present in human serum can impair purR mutant clumping, we hypothesised that mice with antibodies recognizing the FnBPs would be protected from hypervirulence associated with purR::ΦNΣ infection. To test this, we vaccinated groups of 12 mice intraperitoneally with either 1 × 108 heat-killed (HK) USA, 1 × 108 HK USAΔfnbAB, or with PBS on day 0, 6 and 13 (Fig. 6a). On day 23, animals in each group were challenged with either live WT USA or USA purR::ΦNΣ bacteria. In groups vaccinated with WT USA, significantly more animals survived challenge with the purR::ΦNΣ strain, when compared to those vaccinated with USAΔfnbAB or the vehicle control (Fig. 6b). Serum from vaccinated animals demonstrated that mice receiving HK WT USA raised antibodies towards S. aureus antigens, including FnbA/B, while those challenged with HK USAΔfnbAB likewise raised antibodies to many antigens, but not to FnbA/B proteins (Fig. 6c), indicating the protective response is indeed due to anti-FnbA/B antibodies.

Vaccination with S. aureus expressing FnbAB is protective against a challenge with a purR mutant. a Vaccination scheme, with six animals per group. b Survival of animals challenged with 1 × 107 CFU of WT or purR::ΦNΣ S. aureus following vaccination, as outlined in a. *p-value < , **p-value < , based on a Mantel-Cox test, as compared to WT vaccinated, purR::ΦNΣ challenged animals. c Whole cell lysate of WT, WT pfnbA or WTΔfnbAB were used for a western blot, with serum from vaccinated animals or a rabbit anti-Fnb serum (far right) used as a primary antibody

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Discussion

Staphylococcus aureus is a prolific pathogen of humans and animals and, with the emergence of multi-drug resistant strains, efforts to uncover novel drug targets and deliverables have intensified. The discovery and characterisation of novel drug targets often necessitates genetic manipulation of the pathogen under investigation, yet during commonly employed mutagenesis procedures off-target mutations can be induced that profoundly influence the pathogenic potential of this bacterium. Here, we found that exposure of S. aureus to stress such as elevated temperatures (i.e. 42 °C during mutagenesis) or an active host immune response can induce loss of function mutations in the purR gene. PurR is a predicted transcriptional regulator of the purine biosynthetic operon as in other bacteria28,29, and expression of the pur operon is elevated in S. aureus purR mutants. The observation that exposure to stress induces purR mutations could suggest that increased purine biosynthesis is of benefit to S. aureus under these conditions. In E. coli exposure to elevated pH (i.e. pH ≥ ) is also reported to induce purR mutations consistent with the notion that de-repression of the pur operon may promote bacterial survival during exposure to certain stresses30. Remarkably, inactivation of purR in S. aureus causes robust bacterial clumping in the presence of the extracellular matrix protein fibronectin, and causes hyper-virulence in a murine model of systemic infection. Interest in purine metabolism in S. aureus has emerged because disruption of purine biosynthetic pathways can yield growth defects and diminish virulence31,32,33, however, the contribution of the PurR regulator to the physiology of S. aureus has not, to our knowledge, been explored. Consistent with reports of reduced S. aureus fitness when purine biosynthesis is disrupted, we find that provision of PurR in trans reverses the enhanced virulence of purR::ΦNΣ S. aureus USA and attenuates infection (see Fig. 5a). Presumably, overexpression of PurR represses the purEKCSQLFMNHD biosynthetic operon, thereby mimicking the effects of a pur gene deletion, which renders S. aureus less able to grow in human blood and during infection34,35,36.

How purine metabolism impacts S. aureus is unclear, but one possibility is that it involves the bacterial stringent response, which could conceivably enhance resistance to stress. In S. aureus the stringent response can be induced upon amino acid starvation and exposure to the antibiotic mupirocin37, and to the cell wall-targeting antibiotic vancomycin38. Interestingly, exposure to mupirocin and activation of the stringent response has been shown to repress expression of multiple Pur proteins such as PurE, PurQ, and PurK and have a more global impact on gene expression39. Contrary to these findings, our RNAseq data revealed that purR::ΦNΣ S. aureus demonstrated upregulation of pur genes, but no global changes in gene expression, arguing that deletion of the purR gene in S. aureus is unlikely to evoke the stringent response.

The hypervirulent phenotype of purR::ΦNΣ S. aureus is dependent on expression of fibronectin binding proteins A and/or B (FnbA/B) and deletion of the tandemly-duplicated fnbAB genes ablates lethality of the purR::ΦNΣ mutant. FnbA/B are known to bind the extracellular matrix proteins fibronectin and fibrinogen, and contribute to the adhesion of wild-type S. aureus to host tissues, to mediate cellular invasion, and promote virulence during infection40,41,42. Our analysis of fnbA and fnbB gene expression reveals that PurR plays a heretofore undescribed role in repressing FnbA/B expression. Our data support the notion that PurR negatively regulates FnBP expression in early log-phase cells and de-repression of FnBP transcription in purR deficient bacteria occurs only transiently, indicating additional regulatory factors also coordinate FnBP expression. Indeed, previous work characterizing FnbA expression has demonstrated that the global regulators of staphylococcal virulence Agr and Sar coordinate FnbA expression43,44. Nevertheless, aberrant regulation of FnbA/B expression in S. aureus purR mutants plays a vital role in the hyper-agglutination and the hypervirulent phenotypes described herein. Furthermore, the importance of carefully regulated FnBP expression during S. aureus infection is also evidenced by experiments where constitutive expression of plasmid encoded fnbA causes lethal S. aureus infection irrespective of purR (Fig. 4f). At present it is unclear what mechanism within the host causes mortality. However, due to the fact that FnBP proteins promote bacterial clumping and/or interaction with host platelets45,46, it is tempting to speculate that thrombus formation may be a contributing factor. Our data also reveal that FnbA/B contribute significantly to colonization of kidney and liver tissue by purR::ΦNΣ S. aureus during systemic infection. This contrasts wild-type USA where FnbA/B expression seems to play a less important role, as deletion of the fnbA/B genes alone did not significantly affect colonization of the murine heart, kidney or liver (see Fig. 4g). Presumably this can be attributed, in part, to the multitude of virulence factors that S. aureus can express and their potential redundancy. The relative differences in the contribution of FnBP proteins to the pathogenesis of wild-type S. aureus USA and in the purR::ΦNΣ strain is also evident from our vaccination study, where immunization of mice with FnbA/B-expressing S. aureus conferred protection against purR::ΦNΣ lethality but was largely without effect on wild-type USA infection.

The differences observed in purR::ΦNΣ bacterial clumping in human versus horse serum prompted our consideration for the existence of anti-staphylococcal antibodies directed to the FnBPs of S. aureus, as has previously been reported47,48. Human sera do contain naturally occurring anti-FnbA/B antibodies and the presence of these antibodies correlates with reduced purR bacterial clumping in human serum (see Fig. 5). Importantly, these data suggest that some of these naturally occurring antibodies neutralize FnBP-Fn interactions, and the presence of these immunoglobulins may confer protection against the potentially severe consequences of infection with S. aureus purR variants that may arise in humans. Several attempts have been made to create an anti-S. aureus vaccine however, to date, no vaccine has successfully protected against human infection49,50,51,52. Despite this, it is conceivable that immunization with certain S. aureus antigens may offer protection against aspects of staphylococcal pathogenesis (e.g. FnbA/B-dependent events) without altogether preventing infection, a notion that is supported by our data. Nevertheless, the pursuit of a vaccine that will protect against S. aureus infection over and above naturally occurring humoral immunity represents an important aim that will likely require a better understanding of human infection by S. aureus.

In summary, we have discovered that mutations in the S. aureus purR gene, arising from exposure of S. aureus to routine genetic manipulation or to the murine infection environment, can profoundly impact the pathogenesis of this bacterium. Hypervirulence associated with strains carrying purR mutations would undoubtedly offset the potentially desired attenuation phenotype associated with the mutation under study, severely confounding the interpretation of results of S. aureus virulence studies. As such, we urge S. aureus investigators to judiciously characterize mutant strains for off-target mutations affecting toxin production (e.g. sae, as previously reported) and purR, the latter easily tested by growing strains in media containing horse serum. The use of next generation sequencing makes identifying these and other off-target mutations straight forward. Our data show that the mechanism of purR mutant hypervirulence occurs via increased expression of fibronectin binding proteins at low cell densities. The detailed in vivo events occurring during infection of mice with S. aureus purR mutants are currently under study in our laboratory.

Methods

Bacterial growth conditions

Bacterial strains and plasmids used in this study are listed in Table 1 and primers are listed in Table 2. E. coli was grown in Luria-Bertani (LB) broth and S. aureus was grown in tryptic soy broth (TSB) at 37 °C, shaken at  rpm, unless otherwise stated. Where appropriate, media were supplemented with erythromycin (3 µg/mL), chloramphenicol (12 µg/mL), lincomycin (10 µg/mL), ampicillin ( µg/mL) or tetracycline (3 µg/mL). Solid media were supplemented with % (w/v) Bacto agar.

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PCR and construct generation

S. aureus strain USA LAC, cured of the kb plasmid that confers antibiotic resistance, was used as the WT strain for mutant generation, unless otherwise stated. For mobilizing transposon insertion mutations into various genetic backgrounds, phage transduction was performed according to standard techniques. Phage lysate was prepared from the donor strain using phage 80α, recipient strains were infected and transductants selected using appropriate antibiotics. Insertions were confirmed by PCR. Markerless deletions were constructed using the pKOR1 system, as previously described53. Briefly, upstream and downstream regions flanking the FnbAB genes were amplified with primers FnbAB Up F and Up R, and FnbAB Down F and Down R, respectively, using Phusion DNA polymerase and recombined into pKOR1. The resulting vector was passaged through RN and subsequently introduced into strains of interest by electroporation. Genomic deletions were confirmed by PCR with primers hybridizing outside of the cloned area of interest. The purE promoter-glucuronidase fusion reporter was synthesised by Integrated DNA Technologies (IDT, Canada), and ligated into pLL pLL29 was transformed into RN containing a plasmid encoding an integrase and later transduced into USA and derivatives54. For complementation with WT purR or fnbA, the full-length genes were amplified using primers PurR F and PurR R and FnbA F and FnbA R, respectively, ligated into pALC and recombinant plasmids transformed into E. coli. Plasmids were then passaged through RN, prior to transformation into the strain of interest. For insertion of fnb promoters into pGYlux, sequences were amplified from the USA genome with primer pairs pGYluxFnbA F and pGYluxFnbA R (for pGY:fnbA) and pGYluxFnbB F and pGYluxFnbB R (for pGY::fnbB), respectively. Constructs were passaged through RN, prior to transformation into the strain of interest.

Clumping assays

For measurement of clumping in serum (horse or human), overnight cultures in TSB were diluted to OD in 2 mL TSB or TSB with 10% (v/v) serum in a 13 mL tube and grown at 37 °C, with shaking at  rpm for  h. Cultures were allowed to sit without shaking for 5 min and the OD of the middle of the culture was determined. The same cultures were imaged live on a brightfield Leica microscope at ×40 magnification.

Fibronectin removal

To remove fibronectin from horse serum, sterile, heat-inactivated horse serum was passaged over a column of gelatin sepharose (GE healthcare) (column bed volume of  mL) at ~1 mL/min and the flow through collected. The column was washed with ~20 mL of phosphate-buffered saline (PBS) and bound fibronectin was eluted with PBS + 4 M urea. The column was re-generated as per manufacturer’s instructions and the run-through from the first purification passaged again. A total of three passages over the column were performed and the fibronectin-free serum was sterilized by passage through a  µm filter. The different run troughs were used at 10% (v/v) in standard clumping assays, as described above.

Electron microscopy

S. aureus strains were grown in TSB or TSB with 10% horse serum for  h, as previously described for clumping assays. The bacteria were then fixed overnight with a modified Karnovsky’s fixative (% glutaraldehyde (v/v) + 2% paraformaldehyde (v/v) in  M cacodylate buffer, pH ). The fixed bacteria were embedded in a 1% (w/v) agarose suspension and post-fixed with 1% (w/v) osmium tetroxide for 2 h, followed by a 2-h en bloc % (w/v) uranyl acetate strain. Samples were then progressively dehydrated with min treatments of increasingly concentrated ethanol solutions (50, 70, 90, 95, % (v/v)). After dehydration, the samples were embedded in Epon-Araldite and ultrathin sections (70 nm) were cut and placed on nickel grids using an Ultracut microtome. The cut samples were surface stained for 15 min with % (w/v) uranyl acetate and viewed with a Phillips transmission electron microscope equipped with a Hamamatsu Orca 2 MPx HRL camera.

Preparation of proteins

For examination of secreted protein profiles, strains were grown to the desired OD in TSB and normalised to OD of , pelleted by centrifugation, and supernatant mixed with % ethanol at a ratio. Samples were incubated at −20 °C for 4–8 h and proteins pelleted at  × g for 30 min at 4 °C. Pellets were re-suspended in th of the original cultured volume in PBS and stored at −20 °C. For whole cell lysate preparation, cells grown to the desired density were pelleted, washed once with PBS, re-suspended in th of the original volume in PBS with  µg lysostaphin and incubated at 37 °C for 1 h. Samples were passaged twice through a Cell-Disruptor (Constant Systems Ltd.) at 34, p.s.i., pelleted at  × g for 10 min and supernatant harvested. For mass spectrometry analysis of bacterial clumps, the purR::ΦΝΣ mutant strain was grown in TSB with 10% (v/v) for 3 h at 37 °C, and clumps allowed to settle at the bottom of the tube. Clumps were washed three times with PBS, dissolved in 1% SDS at 55 °C for 1 h and run on a 7% SDS polyacrylamide gel. Bands of interest were picked for LC-MS-MS analysis.

Western blots

Strains used for Western blot analysis were the same as described above, with the additional deletion of protein A (spa) and sbi, to eliminate non-specific IgG interactions. Whole cell lysate was prepared as described above, mixed with 1× Laemmli buffer (60 mM Tris-HCl, pH , 2% (w/v) SDS, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, % (w/v) bromphenol blue), boiled for 10 min and separated on a 10% polyacrylamide gel. Following electrophoresis, proteins were transferred to a nitrocellulose membrane following standard protocols. Human, mouse or horse sera ( dilution) or rabbit anti-FnbA antiserum55 ( dilution) were used as a primary antibody, and secondary antibody (conjugated to IRDye ; Li-Cor Biosciences, Lincoln, NE) was used at a , dilution. Membranes were scanned on a Li-Cor Odyssey Infrared Imager (Li-Cor Biosciences) and visualized using Odyssey Version software.

Biofilm assay

Biofilm assays were performed as described previously56. Briefly,  µL of TSB supplemented with % w/v glucose was inoculated with a dilution of an overnight culture. After static incubation at 37 °C for 16–22 h, cells were washed three times with PBS and fixed by drying at 42 °C. Crystal violet (% (w/v)) was used to stain cells for 15 min, before being dissolved in glacial acetic acid (10% (v/v)) and level of adhesion quantified by absorbance at  nm. Absorbance was normalized to the WT strain, which was set to 1.

Luciferase-based measurements of fnb promoter activity

WT or purR::ΦNΣ strains carrying pGYlux constructs with the promoter of fnbA (pGY::fnbA) or fnbB (pGY::fnbB) were used. For luciferase measurements, overnight cultures grown in TSB with chloramphenicol were diluted to OD of in TSB with chloramphenicol and  µl added to a white optical well plate (Thermo Fisher). Growth and luminescence were measured in a BioTek Synergy H4 plate reader at 37 °C with shaking. Data for both absorbance and luminescence was normalised to blank measurements for each time point.

RNA extraction and RNAseq

S. aureus strains were grown overnight, subcultured to an OD equivalent of in TSB and grown to the desired growth phase. Cells equating to an OD of were harvested for each culture, and RNA extraction was performed by E.Z.N.A® total RNA kit (Bio-Rad) according to the manufacturer’s instructions with the addition of  μg/mL lysostaphin to the lysis solution. RNA purity was determined by visualisation on an agarose gel, and RNA concentration was determined by NanoDrop® ND UV-Vis spectrophotometer. cDNA preparation was performed using  ng of total cellular RNA reverse-transcribed using SuperscriptTM II reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. For each qPCR, 1 μg of cDNA was amplified in a Rotor-Gene (Corbett Life Science) using the iScript One-Step RT-PCR kit with SYBR Green (Bio-Rad). Gene expression for each sample was quantified in relation to rpoB expression. A standard curve was generated for each gene examined. For RNAseq, RNA was extracted as above, a library was constructed using an Illumina Script Seq RNA sequencing kit and sequenced on an Illumina MiSeq.

Genome sequence analysis

The nucleotide sequence of the purR gene was downloaded from S. aureus available genome sequences or assemblies from the NCBI database (1st Dec ). Sequences were translated and amino acids aligned to the USA LAC purR sequence using MEGA 7. For strains with sequence changes as compared to USA FPR, the information available was compiled into Supplementary Data 1.

Ethics statements

Human blood was obtained from healthy adult volunteers, with written permission and in compliance with protocol approved by the Office of Research Ethics at the University of Western Ontario. All animal experiments were performed in compliance with guidelines set out by the Canadian Council on Animal Care. All animal protocols (protocol ) were reviewed and approved by the University of Western Ontario Animal Use Subcommittee, a subcommittee of the University Council on Animal Care.

Human serum antibody removal

Blood was allowed to clot for 30 min at RT, centrifuged at  × g for 10 min (no brake) and serum harvested. Serum was filtered through a  µm filter and heat-inactivated for 1 h at 56 °C. For removal of antibody, 4 mL serum was loaded on a HiTrap protein A column (GE healthcare) at 1 mL/min, followed by a 15 min wash (20 mM sodium phosphate, pH 7) at 1 mL/min. Antibody was eluted ( M sodium citrate, pH 4) in three fractions ( mL/each) at 1 mL/min. Serum used for clumping assays was passaged through the column twice. Eluted IgG was filtered through a  µm filter, concentrated with an Amicon ultra centrifugal filter and added to clumping assays containing 10% (v/v) horse serum.

Mouse infections

Six- to eight-week-old female BALB/c mice (Charles River laboratories) were injected via tail vein with  µL of bacterial culture, containing 1 × 106–1 × 107 CFU of bacteria, as described in the text. To prepare the bacteria, strains were grown to OD 2– in TSB, washed twice with PBS and re-suspended to the desired OD in PBS. Infections were allowed to proceed for up to 96 h before animals were euthanized, or when they met guidelines for early euthanasia. Organs were harvested in PBS + % Triton X (Sigma), homogenised in a Bullet Blender Storm (Next Advance, Troy, NY), using two runs of 5 min at setting 10, and metal beads. Dilutions of organ homogenates were plated on TSA for CFU enumeration. For vaccination studies, bacteria were grown to OD of ~, bacteria washed as above, heat killed at 85 °C for 15 min and  µL, equivalent to ~1 × 108 CFU, were injected intraperitoneally (IP). For challenge post vaccination, infections were as outlined above.

Statistical analysis

Statistical analyses were performed with GraphPad Prism software v or v

Reporting Summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this Article.

Data availability

A Reporting Summary for this Article is available as a Supplementary Information file. The source data underlying Figs. 1b, 2b-d, 3c-g, 4c-d, 4g, 5a-c, Supplementary Figures 1 b-d, 2 a-d, 4d and 5 are provided as a Source Data file. Whole genome sequence data have been deposited in the NCBI genbank nucleotide database under accession number PRJNA RNAseq data has been deposited in the NCBI GEO database under accession number GSE Other relevant data are available from the corresponding author upon request.

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Acknowledgements

The project was supported by an operating grant, to D.E.H., from the Canadian Institutes of Health Research. We thank Dr Stephen Tuffs for helpful discussions.

Author information

Author notes
  1. Jessica R. Sheldon

    Present address: Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

  2. These authors contributed equally: Mariya I. Goncheva, Ronald S. Flannagan.

Affiliations

  1. Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada, N6A 5C1

    Mariya I. Goncheva, Ronald S. Flannagan, Brigid E. Sterling, Holly A. Laakso, Nancy C. Friedrich, Julienne C. Kaiser, David W. Watson, Christy H. Wilson, Jessica R. Sheldon, Martin J. McGavin & David E. Heinrichs

  2. Department of Pathology, University of Western Ontario, London, ON, Canada, N6A 5C1

    Patti K. Kiser

Sours: https://www.nature.com/articles/sx

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How dare you behave like that to a defenseless woman. Dear Mrs Leslie, I beg you, forgive my liberty and calm down. I am very grateful to you for a cup of this amazing drink, which has caused an exuberant surge of energy.



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