Faculty Bibliography

The paucity of blood granulocyte populations such as neutrophils in laboratory mice is a notable difference between this model organism and humans, but the cause of this species-specific difference is unclear. We previously demonstrated that laboratory mice released into a seminatural environment, referred to as rewilding, display an increase in blood granulocytes that is associated with expansion of fungi in the gut microbiota. Here, we find that tonic signals from fungal colonization induce sustained granulopoiesis through a mechanism distinct from emergency granulopoiesis, leading to a prolonged expansion of circulating neutrophils that promotes immunity. Fungal colonization after either rewilding or oral inoculation of laboratory mice with Candida albicans induced persistent expansion of myeloid progenitors in the bone marrow. This increase in granulopoiesis conferred greater long-term protection from bloodstream infection by gram-positive bacteria than by the trained immune response evoked by transient exposure to the fungal cell wall component β-glucan. Consequently, introducing fungi into laboratory mice may restore aspects of leukocyte development and provide a better model for humans and free-living mammals that are constantly exposed to environmental fungi.

Extracellular DNase DNASE1L3 maintains tolerance to self-DNA in humans and mice, whereas the role of its homolog DNASE1 remains controversial, and the overall function of secreted DNases in immunity is unclear. We report that deletion of murine DNASE1 neither caused autoreactivity in isolation nor exacerbated lupus-like disease in DNASE1L3-deficient mice. However, combined deficiency of DNASE1 and DNASE1L3 rendered mice susceptible to bloodstream infection with Staphylococcus aureus. DNASE1/DNASE1L3 double-deficient mice mounted a normal innate response to S. aureus and did not accumulate neutrophil extracellular traps (NETs). However, their kidneys manifested severe pathology, increased bacterial burden, and biofilm-like bacterial lesions that contained bacterial DNA and excluded neutrophils. Furthermore, systemic administration of recombinant DNASE1 protein during S. aureus infection rescued the mortality of DNase-deficient mice and ameliorated the disease in wild-type mice. Thus, DNASE1 and DNASE1L3 jointly facilitate the control of bacterial infection by digesting extracellular microbial DNA in biofilms, suggesting the original evolutionary function of secreted DNases as antimicrobial agents.

Background: Escherichia coli and Klebsiella pneumoniae with a piperacillin-tazobactam-nonsusceptible/ceftriaxone-susceptible (TZP-NS/CRO-S) phenotype have been increasingly identified, with limited available literature evaluating treatment strategies. Methods: This was a retrospective study of noncritically ill adults hospitalized between 2013 and 2021 and treated at least 48 hours for TZP-NS/CRO-S E coli or K pneumoniae infections. The primary composite endpoint included escalation to intensive care unit, infection- or treatment-related readmission, mortality, and infection recurrence. Outcomes were compared between groups who received carbapenem (CG) versus carbapenem-sparing agents (CSG) as targeted gram-negative therapy. Results: Of 1062 patients screened, 200 were included (CG, n = 51; CSG, n = 149). Baseline characteristics, including Charlson Comorbidity Index (CCI; median [interquartile range], 6 [3-9] vs 6 [4-9]; P =. 704), were similar between groups, except for more immunocompromised CG patients (29% vs 11%, P =. 001). The most common infection sources were urinary (31% vs 57%, P =. 002) and bloodstream (18% vs 17%, P =. 887). Eighty-eight percent of the CG received meropenem, while 58% of the CSG received ceftriaxone as targeted therapy. There was no statistical difference in the primary endpoint between overall groups (27% vs 17%, P =. 123), nor when stratified by infection source. More patients in the CSG switched to oral therapy (15 [29%] vs 100 [67%], P <. 001). In multivariate analysis, CCI was an independent predictor of the primary outcome (odds ratio [OR], 1.199 [95% confidence interval, 1.074-1.340]; P =. 001), while treatment with carbapenem-sparing therapy was not. Conclusions: Our study did not find improved clinical outcomes with targeted carbapenem therapy for TZP-NS/CRO-S infections. Carbapenem-sparing agents may be considered to spare carbapenems in noncritically ill patients similar to those included in our cohort.

BACKGROUND/UNASSIGNED: METHODS/UNASSIGNED:infections. The primary composite endpoint included escalation to intensive care unit, infection- or treatment-related readmission, mortality, and infection recurrence. Outcomes were compared between groups who received carbapenem (CG) versus carbapenem-sparing agents (CSG) as targeted gram-negative therapy. RESULTS/UNASSIGNED:= .001), while treatment with carbapenem-sparing therapy was not. CONCLUSIONS/UNASSIGNED:Our study did not find improved clinical outcomes with targeted carbapenem therapy for TZP-NS/CRO-S infections. Carbapenem-sparing agents may be considered to spare carbapenems in noncritically ill patients similar to those included in our cohort.

Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.

Staphylococcus aureus is a successful pathogen that produces a wide range of virulence factors that it uses to subvert and suppress the immune system. These include the bicomponent pore-forming leukocidins. How the expression of these toxins is regulated is not completely understood. Here, we describe a screen to identify transcription factors involved in the regulation of leukocidins. The most prominent discovery from this screen is that SarS, a known transcription factor which had previously been described as a repressor of alpha-toxin expression, was found to be a potent repressor of leukocidins LukED and LukSF-PV. We found that inactivating sarS resulted in increased virulence both in an ex vivo model using primary human neutrophils and in an in vivo infection model in mice. Further experimentation revealed that SarS represses leukocidins by serving as an activator of Rot, a critical repressor of toxins, as well as by directly binding and repressing the leukocidin promoters. By studying contemporary clinical isolates, we identified naturally occurring mutations in the sarS promoter that resulted in overexpression of sarS and increased repression of leukocidins in USA300 bloodstream clinical isolates. Overall, these data establish SarS as an important repressor of leukocidins and expand our understanding of how these virulence factors are being regulated in vitro and in vivo by S. aureus.

The regulation of membrane protein activity for cellular functions is critically dependent on the composition of phospholipid membranes. Cardiolipin, a unique phospholipid found in bacterial membranes and mitochondrial membranes of eukaryotes, plays a crucial role in stabilizing membrane proteins and maintaining their function. In the human pathogen Staphylococcus aureus, the SaeRS two-component system (TCS) controls the expression of key virulence factors essential for the bacterium's virulence. The SaeS sensor kinase activates the SaeR response regulator via phosphoryl transfer to bind its gene target promoters. In this study, we report that cardiolipin is critical for sustaining the full activity of SaeRS and other TCSs in S. aureus. The sensor kinase protein SaeS binds directly to cardiolipin and phosphatidylglycerol, enabling SaeS activity. Elimination of cardiolipin from the membrane reduces SaeS kinase activity, indicating that bacterial cardiolipin is necessary for modulating the kinase activities of SaeS and other sensor kinases during infection. Moreover, the deletion of cardiolipin synthase genes cls1 and cls2 leads to reduced cytotoxicity to human neutrophils and lower virulence in a mouse model of infection. These findings suggest a model where cardiolipin modulates the kinase activity of SaeS and other sensor kinases after infection to adapt to the hostile environment of the host and expand our knowledge of how phospholipids contribute to membrane protein function.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

The epidemic community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 lineage has recently become a leading cause of hospital-associated bloodstream infections (BSIs). Here, we leveraged this recent introduction into hospitals and the limited genetic variation across USA300 isolates to identify mutations that contribute to its success in a new environment. We found that USA300 BSI isolates exhibit altered virulence regulation. Using comparative genomics to delineate the genes involved in this phenotype, we discovered repeated and independent mutations in the transcriptional regulator sarZ. Mutations in sarZ resulted in increased virulence of USA300 BSI isolates in a murine model of BSI. The sarZ mutations derepressed the expression and production of the surface protein ClfB, which was critical for the pathogenesis of USA300 BSI isolates. Altogether, these findings highlight ongoing evolution of a major MRSA lineage and suggest USA300 strains can optimize their fitness through altered regulation of virulence.

During the start of the COVID-19 pandemic, shortages of personal protective equipment (PPE) necessitated unprecedented and non-validated approaches to conserve PPE at healthcare facilities, especially in high income countries where single-use disposable PPE was ubiquitous. Our team conducted a systematic literature review to evaluate historic approaches for conserving single-use PPE, expecting that lower-income countries or developing contexts may already be uniquely conserving PPE. However, of the 50 included studies, only 3 originated from middle-income countries and none originated from low-income countries. Data from the included studies suggest PPE remained effective with extended use and with multiple or repeated use in clinical settings, as long as donning and doffing were performed in a standard manner. Multiple decontamination techniques were effective in disinfecting single use PPE for repeated use. These findings can inform healthcare facilities and providers in establishing protocols for safe conservation of PPE supplies and updating existing protocols to improve sustainability and overall resilience. Future studies should evaluate conservation practices in low-resource settings during non-pandemic times to develop strategies for more sustainable and resilient healthcare worldwide.