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Catheter-associated urinary tract infections (CAUTIs), a common cause of healthcare-associated infections, are caused by a diverse array of pathogens that are increasingly becoming antibiotic resistant. We analyze the microbial occurrences in catheter and urine samples from 55 human long-term catheterized patients collected over one year. Although most of these patients were prescribed antibiotics over several collection periods, their catheter samples remain colonized by one or more bacterial species. Examination of a total of 366 catheter and urine samples identify 13 positive and 13 negative genus co-occurrences over 12 collection periods, representing associations that occur more or less frequently than expected by chance. We find that for many patients, the microbial species composition between collection periods is similar. In a subset of patients, we find that the most frequently sampled bacteria, Escherichia coli and Enterococcus faecalis , co-localize on catheter samples. Further, co-culture of paired isolates recovered from the same patients reveals that E. coli significantly augments E. faecalis growth in an artificial urine medium, where E. faecalis monoculture grows poorly. These findings suggest novel strategies to collapse polymicrobial CAUTI in long-term catheterized patients by targeting mechanisms that promote positive co-associations. The authors examine temporal polymicrobial community composition in patients with long-term urinary catheters to identify species co-occurrences and demonstrate uropathogenic Escherichia coli augments growth of a prevalent opportunistic uropathogen in urine.

Microbial pathogens balance growth against tissue damage to achieve maximum fitness. Central carbon metabolism is connected to growth, but how it influences growth/damage balance is largely unknown. Here we examined how carbon flux through the exclusively fermentative metabolism of the pathogenic lactic acid bacterium Streptococcus pyogenes impacts patterns of growth and tissue damage. Using a murine model of soft tissue infection, we systematically examined single and pair-wise mutants that constrained carbon flux through the three major pathways that S . pyogenes employs for reduction of the glycolytic intermediate pyruvate, revealing distinct disease outcomes. Its canonical lactic acid pathway (via lactate dehydrogenase) made a minimal contribution to virulence. In contrast, its two parallel pathways for mixed-acid fermentation played important, but non-overlapping roles. Anaerobic mixed acid fermentation (via pyruvate formate lyase) was required for growth in tissue, while aerobic mixed-acid pathway (via pyruvate dehydrogenase) was not required for growth, but instead regulated levels of tissue damage. Infection of macrophages in vitro revealed that pyruvate dehydrogenase was required to prevent phagolysosomal acidification, which altered expression of the immunosuppressive cytokine IL-10. Infection of IL-10 deficient mice confirmed that the ability of aerobic metabolism to regulate levels of IL-10 plays a key role in the ability of S . pyogenes to modulate levels of tissue damage. Taken together, these results show critical non-overlapping roles for anaerobic and aerobic metabolism in soft tissue infection and provide a mechanism for how oxygen and carbon flux act coordinately to regulate growth/damage balance. Therapies targeting carbon flux could be developed to mitigate tissue damage during severe S . pyogenes infection.

Disease tolerance is a host response to infection that limits collateral damage to host tissues while having a neutral effect on pathogen fitness. Previously, we found that the pathogenic lactic acid bacterium Streptococcus pyogenes manipulates disease tolerance using its aerobic mixed-acid fermentation pathway via the enzyme pyruvate dehydrogenase, but the microbe-derived molecules that mediate communication with the host’s disease tolerance pathways remain elusive. Here we show in a murine model that aerobic mixed-acid fermentation inhibits the accumulation of inflammatory cells including neutrophils and macrophages, reduces the immunosuppressive cytokine interleukin-10, and delays bacterial clearance and wound healing. In infected macrophages, the aerobic mixed-acid fermentation end-products acetate and formate from streptococcal upregulate host acetyl-CoA metabolism and reduce interleukin-10 expression. Inhibiting aerobic mixed-acid fermentation using a bacterial-specific pyruvate dehydrogenase inhibitor reduces tissue damage during murine infection, correlating with increased interleukin-10 expression. Our results thus suggest that reprogramming carbon flow provides a therapeutic strategy to mitigate tissue damage during infection. Disease tolerance helps avoid inflammatory damages when immune system is trying to clear infection, but the mechanisms are still unclear. Here the authors show that the bacteria tap into disease tolerance by altering host cell acetyl-CoA metabolism to suppress innate cell function and cytokine production.

To achieve maximum fitness, pathogens must balance growth with tissue damage, coordinating metabolism and virulence factor expression. In the gram-positive bacterium Streptococcus pyogenes , the DNA-binding transcriptional regulator Carbon Catabolite Protein A (CcpA) is a master regulator of both carbon catabolite repression and virulence, suggesting it coordinates growth/damage balance. To examine this, two murine models were used to compare the virulence of a mutant lacking CcpA with a mutant expressing CcpA locked into its high-affinity DNA-binding conformation (CcpA T307Y ). In models of acute soft tissue infection and of long-term asymptomatic mucosal colonization, both CcpA mutants displayed altered virulence, albeit with distinct growth/damage profiles. Loss of CcpA resulted in a diminished ability to grow in tissue, leading to less damage and early clearance. In contrast, constitutive DNA-binding activity uncoupled the growth/damage relationship, such that high tissue burdens and extended time of carriage were achieved, despite reduced tissue damage. These data demonstrate that growth/damage balance can be actively controlled by the pathogen and implicate CcpA as a master regulator of this relationship. This suggests a model where the topology of the S. pyogenes virulence network has evolved to couple carbon source selection with growth/damage balance, which may differentially influence pathogenesis at distinct tissues.

Catheter-associated urinary tract infections (CAUTIs) are the most prevalent healthcare-associated infection globally, with Enterococcus faecalis posing a significant threat due to widespread antibiotic resistance. This study identifies host cysteine proteases—particularly cathepsin L and caspase-1—as unrecognized drivers of CAUTI pathogenesis and renal fibrosis. Pharmacologic inhibition of these proteases using E64 reduces bladder inflammation, epithelial disruption, and kidney abscesses, while restoring fibrinogen and collagen homeostasis. Strikingly, E64 treatment unmasks a protective eosinophil response via CCR3 signaling that enhances bacterial clearance. Genetic deletion of cathepsin L recapitulates these protective effects, establishing it as a key host factor in E. faecalis persistence. These findings reveal host cysteine proteases as viable therapeutic targets for CAUTI and provide proof-of-concept for host-directed strategies that bypass antibiotic resistance.

Catheter-associated urinary tract infections (CAUTIs) are amongst the most common nosocomial infections worldwide and are difficult to treat partly due to development of multidrug-resistance from CAUTI-related pathogens. Importantly, CAUTI often leads to secondary bloodstream infections and death. A major challenge is to predict when patients will develop CAUTIs and which populations are at-risk for bloodstream infections. Catheter-induced inflammation promotes fibrinogen (Fg) and fibrin accumulation in the bladder which are exploited as a biofilm formation platform by CAUTI pathogens. Using our established mouse model of CAUTI, here we identified that host populations exhibiting either genetic or acquired fibrinolytic-deficiencies, inducing fibrin deposition in the catheterized bladder, are predisposed to severe CAUTI and septicemia by diverse uropathogens in mono- and poly-microbial infections. Furthermore, here we found that Enterococcus faecalis , a prevalent CAUTI pathogen, uses the secreted protease, SprE, to induce fibrin accumulation and create a niche ideal for growth, biofilm formation, and persistence during CAUTI. Catheter-associated urinary tract infections can often lead to secondary bloodstream infections, and catheter-induced bladder inflammation. In this work, authors utilise murine models to probe defective fibrinolysis drives extravascular fibrin formation, potentially predisposing hosts to severe CAUTI.

Virulence factor secretion and assembly occurs at spatially restricted foci in some Gram-positive bacteria. Given the essentiality of the general secretion pathway in bacteria and the contribution of virulence factors to disease progression, the foci that coordinate these processes are attractive antimicrobial targets. In this study, we show in Enterococcus faecalis that SecA and Sortase A, required for the attachment of virulence factors to the cell wall, localize to discrete domains near the septum or nascent septal site as the bacteria proceed through the cell cycle. We also demonstrate that cationic human β-defensins interact with E. faecalis at discrete septal foci, and this exposure disrupts sites of localized secretion and sorting. Modification of anionic lipids by multiple peptide resistance factor, a protein that confers antimicrobial peptide resistance by electrostatic repulsion, renders E. faecalis more resistant to killing by defensins and less susceptible to focal targeting by the cationic antimicrobial peptides. These data suggest a paradigm in which focal targeting by antimicrobial peptides is linked to their killing efficiency and to disruption of virulence factor assembly.

Virulence factors are often multifunctional and contribute to pathogenesis through synergistic mechanisms. For the human pathogen Streptococcus pyogenes , two factors that act synergistically are the S. pyogenes NAD + glycohydrolase (SPN) and streptolysin O (SLO). Through distinct mechanisms, SLO forms pores in host cell membranes and translocates SPN into the host cell cytosol. Two natural variants of SPN exist, one that exhibits NADase activity and one that lacks this function, and both versions are translocated and act in concert with SLO to cause an accelerated death response in epithelial cells. While NADase + SPN is known to trigger a metabolic form of necrosis through the depletion of NAD + , the mechanism by which NADase − SPN induces cell death was unknown. In the studies described here, we examined the pathway of NADase − cell death through analysis of activation patterns of mitogen-activated protein kinases (MAPKs). S. pyogenes infection resulted in activation of members of three MAPK subfamilies (p38, ERK, and JNK). However, only JNK was activated in an SLO-specific manner. NADase − SPN induced necrosis in HeLa epithelial cells associated with depolarization of mitochondrial membranes, activation of NF-κB, and the generation of reactive oxygen species. Remarkably, RNA interference (RNAi) silencing of JNK protected cells from NADase − -SPN-mediated necrosis, suggesting that NADase − SPN triggers a form of programmed necrosis dependent on JNK signaling. Taken together, these data demonstrate that SPN acts with SLO to elicit necrosis through two different mechanisms depending on its NADase activity, i.e., metabolic (NADase + ) or programmed (NADase − ), leading to distinct inflammatory profiles. IMPORTANCE Many bacterial pathogens produce toxins that alter how infected host cells interact with the immune system. For Streptococcus pyogenes , two toxins, a NAD + glycohydrolase (SPN) and streptolysin O (SLO), act in combination to cause infected cells to die. However, there are two natural forms of SPN, and these variants cause dying cells to produce different types of signaling molecules. The NADase + form of SPN kills cells by depleting reserves of NAD + and cellular energy. The other form of SPN lacks this activity (NADase − ); thus, the mechanism by which this variant induces toxicity was unknown. Here, we show that infected cells recognize NADase − SPN through a specific signaling molecule called JNK, which causes these cells to undergo a form of cellular suicide known as programmed necrosis. This helps us to understand how different forms of toxins alter host cell signaling to help S. pyogenes cause different types of diseases. Many bacterial pathogens produce toxins that alter how infected host cells interact with the immune system. For Streptococcus pyogenes , two toxins, a NAD + glycohydrolase (SPN) and streptolysin O (SLO), act in combination to cause infected cells to die. However, there are two natural forms of SPN, and these variants cause dying cells to produce different types of signaling molecules. The NADase + form of SPN kills cells by depleting reserves of NAD + and cellular energy. The other form of SPN lacks this activity (NADase − ); thus, the mechanism by which this variant induces toxicity was unknown. Here, we show that infected cells recognize NADase − SPN through a specific signaling molecule called JNK, which causes these cells to undergo a form of cellular suicide known as programmed necrosis. This helps us to understand how different forms of toxins alter host cell signaling to help S. pyogenes cause different types of diseases.

Gram-positive bacteria in the genus Enterococcus are a frequent cause of catheter-associated urinary tract infection (CAUTI), a disease whose treatment is increasingly challenged by multiantibiotic-resistant strains. We have recently shown that E. faecalis uses the Ebp pilus, a heteropolymeric surface fiber, to bind the host protein fibrinogen as a critical step in CAUTI pathogenesis. Fibrinogen is deposited on catheters due to catheter-induced inflammation and is recognized by the N-terminal domain of EbpA (EbpA NTD ), the Ebp pilus’s adhesin. In a murine model, vaccination with EbpA NTD confers significant protection against CAUTI. Here, we explored the mechanism of protection using passive transfer of immune sera to show that antisera blocking EbpA NTD -fibrinogen interactions not only is prophylactic but also can act therapeutically to reduce bacterial titers of an existing infection. Analysis of 55 clinical CAUTI, bloodstream, and gastrointestinal isolates, including E. faecalis , E. faecium , and vancomycin-resistant enterococci (VRE), revealed a diversity of levels of EbpA expression and fibrinogen-binding efficiency in vitro . Strikingly, analysis of 10 strains representative of fibrinogen-binding diversity demonstrated that, irrespective of EbpA levels, EbpA NTD antibodies were universally protective. The results indicate that, despite diversity in levels of fibrinogen binding, strategies that target the disruption of EbpA NTD -fibrinogen interactions have considerable promise for treatment of CAUTI. IMPORTANCE Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections. Urinary catheterization is a routine medical procedure, and it has been estimated that 30 million Foley catheters are used annually in the United States. Importantly, placement of a urinary catheter renders the patient susceptible to developing a catheter-associated urinary tract infection, accounting for 1 million cases per year. Additionally, these infections can lead to serious complications, including bloodstream infection and death. Enterococcus strains are a common cause of these infections, and management of enterococcal infections has been more difficult in recent years due to the development of antibiotic resistance and the ability of strains to disseminate, resulting in a major threat in hospital settings. In this study, we developed an antibiotic-sparing treatment that is effective against diverse enterococcal isolates, including vancomycin-resistant enterococci, during catheter-associated urinary tract infections.

SpxA is a unique transcriptional regulator highly conserved among members of the phylum Firmicutes that binds RNA polymerase and can act as an antiactivator. Why some Firmicutes members have two highly similar SpxA paralogs is not understood. Here, we show that the SpxA paralogs of the pathogen Streptococcus pyogenes , SpxA1 and SpxA2, act coordinately to regulate virulence by fine-tuning toxin expression and stress resistance. Construction and analysis of mutants revealed that SpxA1 − mutants were defective for growth under aerobic conditions, while SpxA2 − mutants had severely attenuated responses to multiple stresses, including thermal and oxidative stresses. SpxA1 − mutants had enhanced resistance to the cationic antimicrobial molecule polymyxin B, while SpxA2 − mutants were more sensitive. In a murine model of soft tissue infection, a SpxA1 − mutant was highly attenuated. In contrast, the highly stress-sensitive SpxA2 − mutant was hypervirulent, exhibiting more extensive tissue damage and a greater bacterial burden than the wild-type strain. SpxA1 − attenuation was associated with reduced expression of several toxins, including the SpeB cysteine protease. In contrast, SpxA2 − hypervirulence correlated with toxin overexpression and could be suppressed to wild-type levels by deletion of speB . These data show that SpxA1 and SpxA2 have opposing roles in virulence and stress resistance, suggesting that they act coordinately to fine-tune toxin expression in response to stress. SpxA2 − hypervirulence also shows that stress resistance is not always essential for S. pyogenes pathogenesis in soft tissue. IMPORTANCE For many pathogens, it is generally assumed that stress resistance is essential for pathogenesis. For Streptococcus pyogenes , environmental stress is also used as a signal to alter toxin expression. The amount of stress likely informs the bacterium of the strength of the host’s defense response, allowing it to adjust its toxin expression to produce the ideal amount of tissue damage, balancing between too little damage, which will result in its elimination, and too much damage, which will debilitate the host. Here we identify components of a genetic circuit involved in stress resistance and toxin expression that has a fine-tuning function in tissue damage. The circuit consists of two versions of the protein SpxA that regulate transcription and are highly similar but have opposing effects on the severity of soft tissue damage. These results will help us understand how virulence is fine-tuned in other pathogens that have two SpxA proteins. For many pathogens, it is generally assumed that stress resistance is essential for pathogenesis. For Streptococcus pyogenes , environmental stress is also used as a signal to alter toxin expression. The amount of stress likely informs the bacterium of the strength of the host’s defense response, allowing it to adjust its toxin expression to produce the ideal amount of tissue damage, balancing between too little damage, which will result in its elimination, and too much damage, which will debilitate the host. Here we identify components of a genetic circuit involved in stress resistance and toxin expression that has a fine-tuning function in tissue damage. The circuit consists of two versions of the protein SpxA that regulate transcription and are highly similar but have opposing effects on the severity of soft tissue damage. These results will help us understand how virulence is fine-tuned in other pathogens that have two SpxA proteins.