Biology subjects

Lesser, C. F.

Publications and source records attributed to Lesser, C. F..

9 recordsLinked to original sources

bioRxiv2025

Shigella OspF blocks rapid p38-dependent priming of the NAIP-NLRC4 inflammasome

The NAIP-NLRC4 inflammasome senses pathogenic bacteria by recognizing the cytosolic presence of bacterial proteins such as flagellin and type III secretion system (T3SS) subunits. In mice, the NAIP-NLRC4 inflammasome provides robust protection against bacterial pathogens that infect intestinal epithelial cells, including the gastrointestinal pathogen Shigella flexneri. By contrast, humans are highly susceptible to Shigella, despite the ability of human NAIP-NLRC4 to robustly detect Shigella T3SS proteins. Why the NAIP-NLRC4 inflammasome protects mice but not humans against Shigella infection remains unclear. We previously found that human THP-1 cells infected with Shigella lose responsiveness to NAIP-NLRC4 stimuli, while retaining sensitivity to other inflammasome agonists. Using mT3Sf, a "minimal Shigella" system, to express individual secreted Shigella effector proteins, we found that the OspF effector specifically suppresses NAIP-NLRC4-dependent cell death during infection. OspF was previously characterized as a phosphothreonine lyase that inactivates p38 and ERK MAP kinases. We found that p38 was critical for rapid priming of NAIP-NLRC4 activity, particularly in cells with low NAIP-NLRC4 expression. Overall, our results provide a mechanism by which Shigella evades inflammasome activation in humans, and describe a new mechanism for rapid priming of the NAIP-NLRC4 inflammasome.

immunology↗

bioRxiv2025

Macrophages orchestrate elimination of Shigella from the intestinal epithelial cell niche via TLR-induced IL-12 and IFN-γ

Bacteria of the genus Shigella replicate in intestinal epithelial cells and cause shigellosis, a severe diarrheal disease that resolves spontaneously in most healthy individuals. During shigellosis, neutrophils are abundantly recruited to the gut, and have long been thought to be central to Shigella control and pathogenesis. However, how shigellosis resolves remains poorly understood due to the longstanding lack of a tractable and physiological animal model. Here, using our newly developed Nlrc4-/-Casp11-/- mouse model of shigellosis, we unexpectedly find no major role for neutrophils in limiting Shigella or in disease pathogenesis. Instead, we uncover an essential role for macrophages in the host control of Shigella. Macrophages respond to Shigella via TLRs to produce IL-12, which then induces IFN-{gamma}, a cytokine that is essential to control Shigella replication in intestinal epithelial cells. Collectively, our findings reshape our understanding of the innate immune response to Shigella.

immunology↗

bioRxiv2024

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37{degrees}C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence. Author SummaryMany pathogens carry extrachromosomal DNA elements known as plasmids, which encode genes that confer bacterial virulence or antimicrobial resistance (AMR). Acquisition of these plasmids by bacteria can lead to the emergence of new pathogenic traits and the spread of AMR, yet the mechanisms by which plasmids are retained in bacterial populations particularly in the absence of selective pressure remain incompletely understood. Here we show that the major bacterial polyadenylase enzyme PAP I, encoded by the pcnB gene, is critical for the human pathogen Yersinia pseudotuberculosis to maintain its native virulence plasmid as well as AMR plasmids. Very little is known about the process of polyadenylation in bacteria, or the post-transcriptional addition of adenosine residues to the 3 end of transcripts. This study represents the first demonstration that PAP I-mediated polyadenylation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2024

TYPE THREE SECRETION SYSTEM-INDUCED MECHANOPORATION OF VACUOLAR MEMBRANES

SO_SCPLOWUMMARYC_SCPLOWEndomembrane breaching is a crucial strategy employed by intracellular pathogens enclosed within vacuoles to access the nutrient-rich cytosol for intracellular replication. While bacteria use various mechanisms to compromise host membranes, the specific processes and factors involved are often unknown. Shigella flexneri, a major human pathogen, accesses the cytosol relying on the Type Three Secretion System (T3SS) and secreted effectors. Using in-cell correlative light and electron microscopy, we tracked the sequential steps of Shigella host cell entry. Moreover, we captured the T3SS, which projects a needle from the bacterial surface, in the process of puncturing holes in the vacuolar membrane. This initial puncture ensures disruption of the vacuolar membrane. Together this introduces the concept of mechanoporation via a bacterial secretion system as a crucial process for bacterial pathogen-induced membrane damage. GO_SCPLOWRAPHICALC_SCPLOW AO_SCPLOWBSTRACTC_SCPLOW O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/613840v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@1669028org.highwire.dtl.DTLVardef@8259cborg.highwire.dtl.DTLVardef@1d522caorg.highwire.dtl.DTLVardef@1df0f2d_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr({Phi}Stx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC({Phi}Stx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC({Phi}Stx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality. Significance StatementEngineered smart microbes that secrete therapeutics are emerging as treatment modalities, particularly for gut-based diseases. With the growing threat of multidrug-resistant infection, non-antibiotic treatments are urgently needed. The gastrointestinal pathogen enterohemorrhagic E coli (EHEC) can cause the potentially lethal hemolytic uremic syndrome, a toxin-driven disease. Given concerns that antibiotics increase toxin release, treatment is largely limited to supportive care. Here, we show that pre-treatment with a commensal E. coli (HS-PROT3EcT) engineered to secrete an antibody that blocks an essential EHEC virulence factor delays the establishment of an EHEC-like infection in mice. This study strongly suggests that smart microbes that deliver payloads that block colonization factors of gut pathogens can be developed as critically needed alternatives to antibiotics for fighting bacterial infections.

microbiology↗

bioRxiv2022

A hierarchy of cell death pathways confers layered resistance to shigellosis in mice

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease driven by bacterial colonization of colonic intestinal epithelial cells. Vertebrates have evolved programmed cell death pathways that sense invasive enteric pathogens and eliminate their intracellular niche. Previously we reported that genetic removal of one such pathway, the NAIP-NLRC4 inflammasome, is sufficient to convert mice from resistant to susceptible to oral Shigella flexneri challenge (Mitchell, Roncaioli et al., 2020). Here, we investigate the protective role of additional cell death pathways during oral mouse Shigella infection. We find that the Caspase-11 inflammasome, which senses Shigella LPS, restricts Shigella colonization of the intestinal epithelium in the absence of NAIP-NLRC4. However, this protection is limited when Shigella expresses OspC3, an effector that antagonizes Caspase-11 activity. TNF, a cytokine that activates Caspase-8-dependent apoptosis, also provides protection from Shigella colonization of the intestinal epithelium, but only in the absence of both NAIP- NLRC4 and Caspase-11. The combined genetic removal of Caspases-1,-11, and -8 renders mice hyper-susceptible to oral Shigella infection. Our findings uncover a layered hierarchy of cell death pathways that limit the ability of an invasive gastrointestinal pathogen to cause disease.

immunology↗

bioRxiv2021

A Shigella type 3 effector protein co-opts host inositol pyrophosphates for activity

The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and dysenteric disease. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes. ImportanceShigella spp. are important human pathogens and one of the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS), and definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB has been shown to contribute to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad.

microbiology↗

bioRxiv2020

NAIP-NLRC4-deficient mice are susceptible to shigellosis

Bacteria of the genus Shigella cause shigellosis, a severe gastrointestinal disease that is a major cause of diarrhea-associated mortality in humans. Shigellosis develops upon oral ingestion of as few as 100 bacteria, but million-fold higher doses fail to cause disease in mice. The lack of a physiologically relevant mouse model of shigellosis has impeded our understanding of this important human disease, but why mice are resistant is unknown. Here we show that in human cells, but not in mice, Shigella evades detection by the NAIP-NLRC4 inflammasome, an immune sensor present in intestinal epithelial cells (IECs). We find that NAIP-NLRC4-deficient mice are highly susceptible to oral Shigella infection and recapitulate the clinical features of human shigellosis, including bacterial replication in IECs and neutrophilic inflammation of the colon. Confirming a role for bacterial replication in IECs in our new model, a Shigella mutant lacking IcsA, a factor required for cell-to-cell spread among IECs, is attenuated in otherwise susceptible NAIP-NLRC4-deficient mice. Although inflammasome-mediated cell death is widely held to promote Shigella infection and pathogenesis, we instead demonstrate that IEC-specific NAIP-NLRC4-induced cell death is sufficient to protect the host from shigellosis. Thus, NAIP-NLRC4-deficient mice are a physiologically relevant and experimentally tractable model for shigellosis. More broadly, our results suggest that the lack of an inflammasome response in IECs may help explain the extreme susceptibility of humans to shigellosis.

microbiology↗

bioRxiv2018

Functional degradation: a mechanism of NLRP1 inflammasome activation by diverse pathogen enzymes

Inflammasomes are multi-protein platforms that initiate innate immunity by recruitment and activation of Caspase-1. The NLRP1B inflammasome is activated upon direct cleavage by the anthrax lethal toxin protease. However, the mechanism by which cleavage results in NLRP1B activation is unknown. Here we find that cleavage results in proteasome-mediated degradation of the N-terminal domains of NLRP1B, liberating a C-terminal fragment that is a potent Caspase-1 activator. Proteasome-mediated degradation of NLRP1B is both necessary and sufficient for NLRP1B activation. Consistent with our new functional degradation model, we identify IpaH7.8, a Shigella flexneri ubiquitin ligase secreted effector, as an enzyme that induces NLRP1B degradation and activation. Our results provide a unified mechanism for NLRP1B activation by diverse pathogen-encoded enzymatic activities.\n\nOne Sentence SummaryTwo distinct pathogen enzymes activate an innate immune sensor called NLRP1B by a mechanism that requires proteasome-mediated degradation of NLRP1B.

immunology↗