Wei Xu

We report the genome sequence of Klebsiella pneumoniae strain MGC01. Whole-genome sequencing generated 9,155,698 high-quality reads, achieving approximately 265-fold coverage. The final assembly comprises 5,159,509 bp with a GC content of 57.55% and 33 contigs. PlasmidFinder identified an IncM1 plasmid replicon, and Kleborate detected the intrinsic β-lactamase gene bla SHV-1. This sequence supports molecular epidemiology and pathogenicity studies.

Diabetes is associated with impaired immune function and increased susceptibility to severe bacterial infections, yet the pathogen-encoded mechanisms that exacerbate disease in this context remain poorly defined. Streptococcus pyogenes (group A Streptococcus [GAS]) causes invasive skin and soft tissue infections that are disproportionately severe in individuals with diabetes, often accompanied by delayed healing, excessive inflammation, and polymicrobial overgrowth. Here, we investigated how the GAS ClpX-dependent regulatory pathway (CDRP), a global virulence regulator, interacts with diabetic immune dysfunction to shape infection outcomes. Using two murine models of type I diabetes, we show that diabetic mice develop more severe and persistent GAS skin infections characterized by increased bacterial burden, exaggerated inflammatory responses, impaired neutrophil recruitment, excessive neutrophil extracellular trap (NET) accumulation, and frequent polymicrobial infections. Deletion of clpX significantly attenuated GAS virulence in both diabetic and non-diabetic hosts; however, the pathogenic consequences of CDRP were markedly amplified in the diabetic environment. In diabetic mice, CDRP promoted sustained inflammation, altered neutrophil behavior, impaired NET clearance, and enhanced tissue damage, leading to delayed resolution of infection. Mechanistically, ClpX-dependent virulence was associated with dysregulated protease-nuclease activity, excessive NET persistence, and defective neutrophil chemotaxis within diabetic lesions. Together, these findings indicate that while ClpX is a core regulator of GAS virulence, its downstream effects are disproportionately deleterious when host immune clearance is compromised. IMPORTANCE: Diabetic patients experience disproportionately severe bacterial infections, yet the microbial mechanisms that exacerbate disease in this immunocompromised context remain incompletely understood. This study demonstrates that the Streptococcus pyogenes ClpX-dependent regulatory pathway, a central regulator of virulence, amplifies tissue damage and inflammatory dysfunction during diabetic skin infection. ClpX-dependent regulation exacerbates disease by intensifying neutrophil dysregulation, excessive NET accumulation, and impaired resolution of infection in an already compromised host environment. These findings underscore the importance of host-pathogen interactions in shaping infection severity and suggest that targeting pathogen regulatory pathways may be particularly effective in settings of immune dysfunction such as diabetes.

Responsible for over 500,000 deaths annually around the world, Streptococcus pyogenes (group A Streptococcus [GAS]) infections have resurged in the post-COVID-19 era due to immune debt and the rise of strains with enhanced adaptive capabilities. The formidable pathogenicity of GAS is fueled by metabolic plasticity that coordinates virulence with niche-specific adaptation. In this minireview, we dissect how GAS functions as a sophisticated metabolic decision-maker, revealing survival strategies of the bacteria that allow persistence and vulnerabilities that can be targeted for therapeutic development. From the oropharynx to the bloodstream, niche-specific carbon sources and availability dictate downstream biosynthetic processes, creating an integrated metabolic network that controls pathogen fitness. Dynamic shifts in central carbon metabolism are orchestrated by an expanded repertoire of global regulators that directly couple nutrient availability to virulence factor expression. The resulting bacterial metabolic byproducts serve as dual-purpose weapons, limiting competition with commensal microbes and reprogramming host cell immune responses. The ability of GAS to fine-tune and couple metabolism to niche-specific survival factors reveals pathogen-specific targets that can be exploited for therapy. We evaluate high-potential therapeutic strategies that aim to disrupt this critical metabolism-virulence nexus. The development of these precision anti-virulence strategies to counter GAS infections is critical in an era of rising antimicrobial resistance.

Streptococcus pyogenes is a human pathogen that causes severe necrotizing soft tissue infections characterized by rapid and extensive tissue destruction. A key challenge in studying these infections is the lack of experimental models that allow independent assessment of bacterial replication and host-mediated tissue damage. This article presents a standardized and reproducible murine subcutaneous model of S. pyogenes necrotizing skin infection designed to overcome this limitation. The protocol describes the preparation of a log-phase bacterial inoculum, subcutaneous infection of mice, and longitudinal monitoring of lesion development. Key steps include sonication of bacterial cultures to standardize chain length, digital imaging of lesions for quantitative analysis of ulcer area, and determination of bacterial burden through tissue homogenization and plating. This versatile model can be readily adapted to evaluate the effects of specific bacterial genes, host factors, or therapeutic interventions on infection progression and tissue pathology. By enabling reproducible and quantitative assessment of both bacterial and host parameters, this method provides a robust platform for studying pathogenesis and testing strategies to limit tissue damage during invasive streptococcal infection.

Streptococcus pyogenes is a human pathogen that causes severe necrotizing soft tissue infections characterized by rapid and extensive tissue destruction. A key challenge in studying these infections is the lack of experimental models that allow independent assessment of bacterial replication and host-mediated tissue damage. This article presents a standardized and reproducible murine subcutaneous model of S. pyogenes necrotizing skin infection designed to overcome this limitation. The protocol describes the preparation of a log-phase bacterial inoculum, subcutaneous infection of mice, and longitudinal monitoring of lesion development. Key steps include sonication of bacterial cultures to standardize chain length, digital imaging of lesions for quantitative analysis of ulcer area, and determination of bacterial burden through tissue homogenization and plating. This versatile model can be readily adapted to evaluate the effects of specific bacterial genes, host factors, or therapeutic interventions on infection progression and tissue pathology. By enabling reproducible and quantitative assessment of both bacterial and host parameters, this method provides a robust platform for studying pathogenesis and testing strategies to limit tissue damage during invasive streptococcal infection.

Building on our finding that Streptococcus pyogenes pyruvate dehydrogenase (PDH) suppresses host disease tolerance (DT) via short-chain fatty acid (SCFA)-mediated modulation of host acetyl-CoA and IL-10 levels, we characterize the global transcriptomic and epigenetic mechanisms underlying this immunometabolic manipulation. Combining new histological and ultrastructural analyses with an in-depth re-analysis of single-cell and bulk RNA-seq datasets to more comprehensively characterize the DT response, we show that PDH deficiency is associated with broad immunologic rewiring, characterized by intracellular bacterial containment within phagocytes, expansion of pro-resolving myeloid cells, and altered cell-cell communication. Metabolic analysis of ΔPdh-infected tissues revealed a shift away from acetyl-CoA metabolism towards glycolysis and the coordinated activation of a multi-faceted DT program, encompassing hypoxia signaling, iron handling, and the NRF2-mediated antioxidant response. Crucially, a focused re-analysis of existing transcriptome datasets from Histone Deacetylase (HDAC)-inhibited macrophages suggested that Trichostatin A (TSA) abrogates the protective transcriptome in ΔPdh infection, indicating that acetylation-dependent repression functions as a key regulator of the host DT response. By integrating new experimental data with advanced computational analyses, our work reveals a bacterial strategy of metabolic-epigenetic crosstalk, suggesting acetylation as a critical control point for mitigating infection-associated tissue damage.

The bacterium Enterococcus faecalis is a leading cause of catheter-associated urinary tract infection (CAUTI), whose treatment is increasingly challenged by antibiotic resistance. In examining alternative therapies, we previously found that the cysteine protease inhibitor E64 dramatically reduced bladder inflammation and bacterial dissemination in a murine model of E. faecalis CAUTI. However, the role of cysteine proteases in pathogenesis and the target(s) of E64 are unknown. Here, we found that while it did not affect E. faecalis growth in vitro , in a murine CAUTI model, E64 reduced host caspase-1-dependent cellular apoptosis and necrosis, levels of several pro-inflammatory cytokines, bladder epithelial damage, and formation of renal abscesses. Analysis of host cell transcription and inflammatory cell populations revealed that E64 did not affect neutrophil numbers but did enhance the expression of C-C chemokine receptor type 3 (CCR3), a receptor for eosinophil-specific chemokines, with a concomitant increase in eosinophil numbers. Treatments that reduced or increased the eosinophil response (anti-interleukin 5 antibody or eotaxin, respectively) confirmed a role for eosinophils in controlling bacterial burdens. Analysis of a panel of host cysteine proteases in vivo demonstrated that E64 decreased the activation of cathepsin L; subsequently, we found that the infection of cathepsin L-deficient mice yielded lower catheter and bladder colonization compared to wild-type mice. In total, we have shown that host cysteine proteases exacerbate CAUTI pathogenesis, suggesting that cysteine protease modulation may represent a novel approach for the treatment of persistent CAUTI. IMPORTANCE: 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.

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.

We have developed GmPcides from a peptidomimetic dihydrothiazolo ring-fused 2-pyridone scaffold that has antimicrobial activities against a broad spectrum of Gram-positive pathogens. Here, we examine the treatment efficacy of GmPcides using skin and soft tissue infection (SSTI) and biofilm formation models by Streptococcus pyogenes . Screening our compound library for minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations identified GmPcide PS757 as highly active against S. pyogenes . Treatment of S. pyogenes biofilm with PS757 revealed robust efficacy against all phases of biofilm formation by preventing initial biofilm development, ceasing biofilm maturation and eradicating mature biofilm. In a murine model of S. pyogenes SSTI, subcutaneous delivery of PS757 resulted in reduced levels of tissue damage, decreased bacterial burdens, and accelerated rates of wound healing, which were associated with down-regulation of key virulence factors, including M protein and the SpeB cysteine protease. These data demonstrate that GmPcides show considerable promise for treating S. pyogenes infections.