The tempo and mode of gene regulatory programs during bacterial infection
Avital, Gal; Kuperwaser, Felicia; Pountain, Andrew W; Lacey, Keenan A; Zwack, Erin E; Podkowik, Magdalena; Shopsin, Bo; Torres, Victor J; Yanai, Itai
Innate immune recognition of bacterial pathogens is a key determinant of the ensuing systemic response, and host or pathogen heterogeneity in this early interaction can impact the course of infection. To gain insight into host response heterogeneity, we investigate macrophage inflammatory dynamics using primary human macrophages infected with Group B Streptococcus. Transcriptomic analysis reveals discrete cellular states within responding macrophages, one of which consists of four sub-states, reflecting inflammatory activation. Infection with six additional bacterial species-Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Yersinia pseudotuberculosis, Shigella flexneri, and Salmonella enterica-recapitulates these states, though at different frequencies. We show that modulating the duration of infection and the presence of a toxin impacts inflammatory trajectory dynamics. We provide evidence for this trajectory in infected macrophages in an inÂ vivo model of Staphylococcus aureus infection. Our cell-state analysis defines a framework for understanding inflammatory activation dynamics in response to bacterial infection.
ACE2-containing defensosomes serve as decoys to inhibit SARS-CoV-2 infection
Ching, Krystal L; de Vries, Maren; Gago, Juan; Dancel-Manning, Kristen; Sall, Joseph; Rice, William J; Barnett, Clea; Khodadadi-Jamayran, Alireza; Tsirigos, Aristotelis; Liang, Feng-Xia; Thorpe, Lorna E; Shopsin, Bo; Segal, Leopoldo N; Dittmann, Meike; Torres, Victor J; Cadwell, Ken
Extracellular vesicles of endosomal origin, exosomes, mediate intercellular communication by transporting substrates with a variety of functions related to tissue homeostasis and disease. Their diagnostic and therapeutic potential has been recognized for diseases such as cancer in which signaling defects are prominent. However, it is unclear to what extent exosomes and their cargo inform the progression of infectious diseases. We recently defined a subset of exosomes termed defensosomes that are mobilized during bacterial infection in a manner dependent on autophagy proteins. Through incorporating protein receptors on their surface, defensosomes mediated host defense by binding and inhibiting pore-forming toxins secreted by bacterial pathogens. Given this capacity to serve as decoys that interfere with surface protein interactions, we investigated the role of defensosomes during infection by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent of Coronavirus Disease 2019 (COVID-19). Consistent with a protective function, exosomes containing high levels of the viral receptor ACE2 in bronchoalveolar lavage fluid (BALF) from critically ill COVID-19 patients was associated with reduced intensive care unit (ICU) and hospitalization times. We found ACE2+ exosomes were induced by SARS-CoV-2 infection and activation of viral sensors in cell culture, which required the autophagy protein ATG16L1, defining these as defensosomes. We further demonstrate that ACE2+ defensosomes directly bind and block viral entry. These findings suggest that defensosomes may contribute to the antiviral response against SARS-CoV-2 and expand our knowledge on the regulation and effects of extracellular vesicles during infection.
Staphylococcus aureus induces a muted host response in human blood that blunts the recruitment of neutrophils
Zwack, Erin E; Chen, Ze; Devlin, Joseph C; Li, Zhi; Zheng, Xuhui; Weinstock, Ada; Lacey, Keenan A; Fisher, Edward A; Fenyö, David; Ruggles, Kelly V; Loke, P'ng; Torres, Victor J
Structural basis for inhibition of the drug efflux pump NorA from Staphylococcus aureus
Brawley, Douglas N; Sauer, David B; Li, Jianping; Zheng, Xuhui; Koide, Akiko; Jedhe, Ganesh S; Suwatthee, Tiffany; Song, Jinmei; Liu, Zheng; Arora, Paramjit S; Koide, Shohei; Torres, Victor J; Wang, Da-Neng; Traaseth, Nathaniel J
Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA-Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.
Microbiome-Independent Effects of Antibiotics in a Murine Model of Nosocomial Infections
Lacey, Keenan A; Gonzalez, Sandra; Yeung, Frank; Putzel, Gregory; Podkowik, Magdalena; Pironti, Alejandro; Shopsin, Bo; Cadwell, Ken; Torres, Victor J
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of hospital-acquired pneumonia. To better manage patients with MRSA pneumonia, we require a greater understanding of the host-pathogen interactions during infection. MRSA research focuses on highly virulent and cytotoxic strains, which demonstrate robust phenotypes in animal models of infection. However, nosocomial infections are often caused by hospital-acquired MRSA (HA-MRSA) isolates that exhibit low cytotoxicity and few or no phenotypes in mice, thereby confounding mechanistic studies of pathogenesis. Consequently, virulence pathways utilized by HA-MRSA in nosocomial pneumonia are largely unknown. Here, we report that conditioning mice with broad-spectrum antibiotics lowers the barrier to pneumonia, thereby transforming otherwise avirulent HA-MRSA isolates into lethal pathogens. HA-MRSA isolates are avirulent in gnotobiotic mice, mimicking results in conventional animals. Thus, the observed enhanced susceptibility to infection in antibiotic-treated mice is not due to depletion of the microbiota. More generally, we found that antibiotic conditioning leads to increased susceptibility to infection by diverse antimicrobial-resistant (AMR) pathogens of low virulence. Treatment with antibiotics leads to dehydration and malnutrition, suggesting a potential role for these clinically relevant and reducible hospital complications in susceptibility to pathogens. In sum, the model described here mitigates the impact of low virulence in immunocompetent mice, providing a convenient model to gain fundamental insight into the pathogenesis of nosocomial pathogens. IMPORTANCE Antimicrobial-resistant (AMR) pathogens are responsible for over 2.8 million infections and over 35,000 deaths per year in the United States. To study these microbes, animal models that are susceptible to these pathogens are required. However, many of these pathogens exhibit low virulence in conventional mice, which has negatively impacted mechanistic studies. Here, we show that mice treated with antibiotics in their drinking water become exquisitely susceptible to low-virulence AMR pathogens. Surprisingly, the increased susceptibility was independent of the impact of antibiotics on the microbiome and seems to be due to an unintended consequence of antibiotic treatment: weight loss due to dehydration and caloric restriction. Unlike other models used to sensitize mice to low-virulence pathogens, our model does not reduce phagocyte numbers. Thus, here, we describe an immunocompetent mouse model to facilitate the identification of novel targets and accelerate the development of preventives and therapeutics to combat infections by AMR pathogens.
Human OTULIN haploinsufficiency impairs cell-intrinsic immunity to staphylococcal Î±-toxin
Spaan, AndrÃ¡s N; Neehus, Anna-Lena; Laplantine, Emmanuel; Staels, Frederik; Ogishi, Masato; Seeleuthner, Yoann; Rapaport, Franck; Lacey, Keenan A; Van Nieuwenhove, Erika; Chrabieh, Maya; Hum, David; Migaud, Mélanie; Izmiryan, Araksya; Lorenzo, Lazaro; Kochetkov, Tatiana; Heesterbeek, Dani A C; Bardoel, Bart W; DuMont, Ashley L; Dobbs, Kerry; Chardonnet, Solenne; Heissel, SÃ¸ren; Baslan, Timour; Zhang, Peng; Yang, Rui; Bogunovic, Dusan; Wunderink, Herman F; Haas, Pieter-Jan A; Molina, Henrik; Van Buggenhout, Griet; Lyonnet, Stanislas; Notarangelo, Luigi D; SeppÃ¤nen, Mikko R J; Weil, Robert; Seminario, Gisela; Gomez-Tello, Héctor; Wouters, Carine; Mesdaghi, Mehrnaz; Shahrooei, Mohammad; Bossuyt, Xavier; Sag, Erdal; Topaloglu, Rezan; Ozen, Seza; Leavis, Helen L; van Eijk, Maarten M J; Bezrodnik, Liliana; Blancas Galicia, Lizbeth; Hovnanian, Alain; Nassif, Aude; Bader-Meunier, Brigitte; Neven, Bénédicte; Meyts, Isabelle; Schrijvers, Rik; Puel, Anne; Bustamante, Jacinta; Aksentijevich, Ivona; Kastner, Daniel L; Torres, Victor J; Humblet-Baron, Stéphanie; Liston, Adrian; Abel, Laurent; Boisson, Bertrand; Casanova, Jean-Laurent
The molecular basis of interindividual clinical variability upon infection with Staphylococcus aureus is unclear. We describe patients with haploinsufficiency for the linear deubiquitinase OTULIN, encoded by a gene on chromosome 5p. Patients suffer from episodes of life-threatening necrosis, typically triggered by S. aureus infection. The disorder is phenocopied in patients with the 5p- (Cri-du-Chat) chromosomal deletion syndrome. OTULIN haploinsufficiency causes an accumulation of linear ubiquitin in dermal fibroblasts, but tumor necrosis factor receptor-mediated nuclear factor ÎºB signaling remains intact. Blood leukocyte subsets are unaffected. The OTULIN-dependent accumulation of caveolin-1 in dermal fibroblasts, but not leukocytes, facilitates the cytotoxic damage inflicted by the staphylococcal virulence factor Î±-toxin. Naturally elicited antibodies against Î±-toxin contribute to incomplete clinical penetrance. Human OTULIN haploinsufficiency underlies life-threatening staphylococcal disease by disrupting cell-intrinsic immunity to Î±-toxin in nonleukocytic cells.
Pathogen Species Is Associated With Mortality in Nosocomial Bloodstream Infection in Patients With COVID-19
Gago, Juan; Filardo, Thomas D; Conderino, Sarah; Magaziner, Samuel J; Dubrovskaya, Yanina; Inglima, Kenneth; Iturrate, Eduardo; Pironti, Alejandro; Schluter, Jonas; Cadwell, Ken; Hochman, Sarah; Li, Huilin; Torres, Victor J; Thorpe, Lorna E; Shopsin, Bo
Background/UNASSIGNED:The epidemiology of nosocomial bloodstream infections (NBSIs) in patients with coronavirus disease 2019 (COVID-19) is poorly understood, due in part to substantial disease heterogeneity resulting from multiple potential pathogens. Methods/UNASSIGNED:We identified risk factors for NBSIs and examined the association between NBSIs and mortality in a retrospective cohort of patients hospitalized with COVID-19 in 2 New York City hospitals during the height of the pandemic. We adjusted for the potential effects of factors likely to confound that association, including age, race, illness severity upon admission, and underlying health status. Results/UNASSIGNED:infections did not have an identifiable source and were not associated with common risk factors for infection by these organisms. Conclusions/UNASSIGNED:Pathogen species and mortality exhibited temporal differences. Early recognition of risk factors among COVID-19 patients could potentially decrease NBSI-associated mortality through early COVID-19 and antimicrobial treatment.
The Major Autolysin Atl Regulates the Virulence of Staphylococcus aureus by Controlling the Sorting of LukAB
Zheng, Xuhui; Ma, Sheya Xiao; St John, Amelia; Torres, Victor J
Infections caused by the Gram-positive bacterium Staphylococcus aureus remain a significant health threat globally. The production of bicomponent pore-forming leukocidins plays an important role in S. aureus pathogenesis. Transcriptionally, these toxins are primarily regulated by the Sae and Agr regulatory systems. However, the posttranslational regulation of these toxins is largely unexplored. In particular, one of the leukocidins, LukAB, has been shown to be both secreted into the extracellular milieu and associated with the bacterial cell envelope. Here, we report that a major cell wall hydrolase, autolysin (Atl), controls the sorting of LukAB from the cell envelope to the extracellular milieu, an effect independent of transcriptional regulation. By influencing the sorting of LukAB, Atl modulates S. aureus cytotoxicity toward primary human neutrophils. Mechanistically, we found that the reduction in peptidoglycan cleavage and increased LukAB secretion in the atl mutant can be reversed through the supplementation of exogenous mutanolysin. Altogether, our study revealed that the cell wall hydrolase activity of Atl and the cleavage of peptidoglycan play an important role in controlling the sorting of S. aureus toxins during secretion.
Vaccination With Detoxified Leukocidin AB Reduces Bacterial Load in a Staphylococcus aureus Minipig Deep Surgical Wound Infection Model
Fernandez, Jeffrey; Sanders, Holly; Henn, Jessica; Wilson, Jolaine M; Malone, Danielle; Buoninfante, Alessandra; Willms, Matthew; Chan, Rita; DuMont, Ashley L; McLahan, Craig; Grubb, Kaitlyn; Romanello, Anthony; van den Dobbelsteen, Germie; Torres, Victor J; Poolman, Jan T
Vaccines against Staphylococcus aureus have eluded researchers for >3 decades while the burden of staphylococcal diseases has increased. Early vaccine attempts mainly used rodents to characterize preclinical efficacy, and all subsequently failed in human clinical efficacy trials. More recently, leukocidin AB (LukAB) has gained interest as a vaccine antigen. We developed a minipig deep surgical wound infection model offering 3 independent efficacy readouts: bacterial load at the superficial and at the deep-seated surgical site, and dissemination of bacteria. Due to similarities with humans, minipigs are an attractive option to study novel vaccine candidates. With this model, we characterized the efficacy of a LukAB toxoid as vaccine candidate. Compared to control animals, a 3-log reduction of bacteria at the deep-seated surgical site was observed in LukAB-treated minipigs and dissemination of bacteria was dramatically reduced. Therefore, LukAB toxoids may be a useful addition to S. aureus vaccines and warrant further study.
Genome-Wide CRISPR-Cas9 Screen Does Not Identify Host Factors Modulating Streptococcus agalactiae Î²-Hemolysin/Cytolysin-Induced Cell Death
Shahi, Ifrah; Llaneras, Cristina N; Perelman, Sofya S; Torres, Victor J; Ratner, Adam J
Pore-forming toxins (PFTs) are commonly produced by pathogenic bacteria, and understanding them is key to the development of virulence-targeted therapies. Streptococcus agalactiae, or group B Streptococcus (GBS), produces several factors that enhance its pathogenicity, including the PFT Î²-hemolysin/cytolysin (Î²hc). Little is understood about the cellular factors involved in Î²hc pore formation. We conducted a whole-genome CRISPR-Cas9 forward genetic screen to identify host genes that might contribute to Î²hc pore formation and cell death. While the screen identified the established receptor, CD59, in control experiments using the toxin intermedilysin (ILY), no clear candidate genes were identified that were required for Î²hc-mediated lethality. Of the top targets from the screen, two genes involved in membrane remodeling and repair represented candidates that might modulate the kinetics of Î²hc-induced cell death. Upon attempted validation of the results using monoclonal cell lines with targeted disruption of these genes, no effect on Î²hc-mediated cell lysis was observed. The CRISPR-Cas9 screen results are consistent with the hypothesis that Î²hc does not require a single nonessential host factor to mediate target cell death. IMPORTANCE CRISPR-Cas9 forward genetic screens have been used to identify host cell targets required by bacterial toxins. They have been used successfully to both verify known targets and elucidate novel host factors required by toxins. Here, we show that this approach fails to identify host factors required for cell death due to Î²hc, a toxin required for GBS virulence. These data suggest that Î²hc may not require a host cell receptor for toxin function or may require a host receptor that is an essential gene and would not be identified using this screening strategy.