Listeria monocytogenes virulence factors, including listeriolysin O, are secreted in biologically active extracellular vesicles.

Outer membrane vesicles produced by Gram-negative bacteria have been studied for half a century but the possibility that Gram-positive bacteria secrete extracellular vesicles (EVs) was not pursued until recently due to the assumption that the thick peptidoglycan cell wall would prevent their release to the environment. However, following their discovery in fungi, which also have cell walls, EVs have now been described for a variety of Gram-positive bacteria. EVs purified from Gram-positive bacteria are implicated in virulence, toxin release, and transference to host cells, eliciting immune responses, and spread of antibiotic resistance. Listeria monocytogenes is a Gram-positive bacterium that causes listeriosis. Here we report that L. monocytogenes produces EVs with diameters ranging from 20 to 200 nm, containing the pore-forming toxin listeriolysin O (LLO) and phosphatidylinositol-specific phospholipase C (PI-PLC). Cell-free EV preparations were toxic to mammalian cells, the murine macrophage cell line J774.16, in a LLO-dependent manner, evidencing EV biological activity. The deletion of plcA increased EV toxicity, suggesting PI-PLC reduced LLO activity. Using simultaneous metabolite, protein, and lipid extraction (MPLEx) multiomics we characterized protein, lipid, and metabolite composition of bacterial cells and secreted EVs and found that EVs carry the majority of listerial virulence proteins. Using immunogold EM we detected LLO at several organelles within infected human epithelial cells and with high-resolution fluorescence imaging we show that dynamic lipid structures are released from L. monocytogenes during infection. Our findings demonstrate that L. monocytogenes uses EVs for toxin release and implicate these structures in mammalian cytotoxicity.

Broad detection of bacterial type III secretion system and flagellin proteins by the human NAIP/NLRC4 inflammasome.

Inflammasomes are cytosolic multiprotein complexes that initiate host defense against bacterial pathogens by activating caspase-1-dependent cytokine secretion and cell death. In mice, specific nucleotide-binding domain, leucine-rich repeat-containing family, apoptosis inhibitory proteins (NAIPs) activate the nucleotide-binding domain, leucine-rich repeat-containing family, CARD domain-containing protein 4 (NLRC4) inflammasome upon sensing components of the type III secretion system (T3SS) and flagellar apparatus. NAIP1 recognizes the T3SS needle protein, NAIP2 recognizes the T3SS inner rod protein, and NAIP5 and NAIP6 recognize flagellin. In contrast, humans encode a single functional NAIP, raising the question of whether human NAIP senses one or multiple bacterial ligands. Previous studies found that human NAIP detects both flagellin and the T3SS needle protein and suggested that the ability to detect both ligands was achieved by multiple isoforms encoded by the single human NAIP gene. Here, we show that human NAIP also senses the Salmonella Typhimurium T3SS inner rod protein PrgJ and that T3SS inner rod proteins from multiple bacterial species are also detected. Furthermore, we show that a single human NAIP isoform is capable of sensing the T3SS inner rod, needle, and flagellin. Our findings indicate that, in contrast to murine NAIPs, promiscuous recognition of multiple bacterial ligands is conferred by a single human NAIP.

Neutrophils and Ly6Chi monocytes collaborate in generating an optimal cytokine response that protects against pulmonary Legionella pneumophila infection.

Early responses mounted by both tissue-resident and recruited innate immune cells are essential for host defense against bacterial pathogens. In particular, both neutrophils and Ly6Chi monocytes are rapidly recruited to sites of infection. While neutrophils and monocytes produce bactericidal molecules, such as reactive nitrogen and oxygen species, both cell types are also capable of synthesizing overlapping sets of cytokines important for host defense. Whether neutrophils and monocytes perform redundant or non-redundant functions in the generation of anti-microbial cytokine responses remains elusive. Here, we sought to define the contributions of neutrophils and Ly6Chi monocytes to cytokine production and host defense during pulmonary infection with Legionella pneumophila, responsible for the severe pneumonia Legionnaires' disease. We found that both neutrophils and monocytes are critical for host defense against L. pneumophila. Both monocytes and neutrophils contribute to maximal IL-12 and IFNγ responses, and monocytes are also required for TNF production. Moreover, natural killer (NK) cells, NKT cells, and γδ T cells are sources of IFNγ, and monocytes direct IFNγ production by these cell types. Thus, neutrophils and monocytes cooperate in eliciting an optimal cytokine response that promotes effective control of bacterial infection.

Hfq and three Hfq-dependent small regulatory RNAs-MgrR, RyhB and McaS-coregulate the locus of enterocyte effacement in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a significant cause of infantile diarrhea and death in developing countries. The pathogenicity island locus of enterocyte effacement (LEE) is essential for EPEC to cause diarrhea. Besides EPEC, the LEE is also present in other gastrointestinal pathogens, most notably enterohemorrhagic E. coli (EHEC). Whereas transcriptional control of the LEE has been meticulously examined, posttranscriptional regulation, including the role of Hfq-dependent small RNAs, remains undercharacterized. However, the past few years have witnessed a surge in the identification of riboregulators of the LEE in EHEC. Contrastingly, the posttranscriptional regulatory landscape of EPEC remains cryptic. Here we demonstrate that the RNA-chaperone Hfq represses the LEE of EPEC by targeting the 5' untranslated leader region of grlR in the grlRA mRNA. Three conserved small regulatory RNAs (sRNAs)-MgrR, RyhB and McaS-are involved in the Hfq-dependent regulation of grlRA MgrR and RyhB exert their effects by directly base-pairing to the 5' region of grlR Whereas MgrR selectively represses grlR but activates grlA, RyhB represses gene expression from the entire grlRA transcript. Meanwhile, McaS appears to target the grlRA mRNA indirectly. Thus, our results provide the first definitive evidence that implicates multiple sRNAs in regulating the LEE and the resulting virulence of EPEC.

Lambda Red-mediated Recombineering in the Attaching and Effacing Pathogen Escherichia albertii.

BACKGROUND: The ability to introduce site-specific mutations in bacterial pathogens is essential towards understanding their molecular mechanisms of pathogenicity. This has been greatly facilitated by the genetic engineering technique of recombineering. In recombineering, linear double- or single-stranded DNA molecules with two terminal homology arms are electroporated into hyperrecombinogenic bacteria that express a phage-encoded recombinase. The recombinase catalyzes the replacement of the endogenous allele with the exogenous allele to generate selectable or screenable recombinants. In particular, lambda red recombinase has been instrumental in engineering mutations to characterize the virulence arsenal of the attaching and effacing (A/E) pathogens enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC), and Citrobacter rodentium. Escherichia albertii is another member of this taxon; however, the virulence of E. albertii remains cryptic despite accumulating evidence that E. albertii is an emerging pathogen. Multiple retrospective studies have reported that a substantial number of EPEC and EHEC isolates (~15 %) that were previously incriminated in human outbreaks actually belong to the E. albertii lineage. Thus, there is increased urgency to reliably identify and rapidly engineer mutations in E. albertii to systematically characterize its virulence determinants. To the best of our knowledge not a single chromosomal gene has been altered by targeted mutagenesis in E. albertii since it was first isolated almost 25 years ago. This is disconcerting because an E. albertii outbreak could cause significant morbidity and mortality owing to our inadequate understanding of its virulence program. RESULTS: In this report we describe a modified lambda red recombineering protocol to mutagenize E. albertii. As proof of principle, we successfully deleted three distinct virulence-associated genetic loci - ler, grlRA, and hfq - and replaced each wild type allele by a mutant allele with an encodable drug resistance cassette bracketed by FRT sites. Subsequently, the FRT-site flanked drug resistance marker was evicted by FLP-dependent site-specific recombination to generate excisants containing a solitary FRT site. CONCLUSIONS: Our protocol will enable researchers to construct marked and unmarked genome-wide mutations in E. albertii, which, in turn, will illuminate its molecular mechanisms of pathogenicity and aid in developing appropriate preventative and therapeutic approaches to combat E. albertii outbreaks.

Discovery of the Collaborative Nature of Science with Undergraduate Science Majors and Non-Science Majors through the Identification of Microorganisms Enriched in Winogradsky Columns.

Today's science classrooms are addressing the need for non-scientists to become scientifically literate. A key aspect includes the recognition of science as a process for discovery. This process relies upon interdisciplinary collaboration. We designed a semester-long collaborative exercise that allows science majors taking a general microbiology course and non-science majors taking an introductory environmental science course to experience collaboration in science by combining their differing skill sets to identify microorganisms enriched in Winogradsky columns. These columns are self-sufficient ecosystems that allow researchers to study bacterial populations under specified environmental conditions. Non-science majors identified phototrophic bacteria enriched in the column by analyzing the signature chlorophyll absorption spectra whereas science majors used 16S rRNA gene sequencing to identify the general bacterial diversity. Students then compiled their results and worked together to generate lab reports with their final conclusions identifying the microorganisms present in their column. Surveys and lab reports were utilized to evaluate the learning objectives of this activity. In pre-surveys, nonmajors' and majors' answers diverged considerably, with majors providing responses that were more accurate and more in line with the working definition of collaboration. In post-surveys, the answers between majors and nonmajors converged, with both groups providing accurate responses. Lab reports showed that students were able to successfully identify bacteria present in the columns. These results demonstrate that laboratory exercises designed to group students across disciplinary lines can be an important tool in promoting science education across disciplines.