Listeria monocytogenes exploits host exocytosis to promote cell-to-cell spread.

The facultative intracellular pathogen Listeria monocytogenes uses an actin-based motility process to spread within human tissues. Filamentous actin from the human cell forms a tail behind bacteria, propelling microbes through the cytoplasm. Motile bacteria remodel the host plasma membrane into protrusions that are internalized by neighboring cells. A critical unresolved question is whether generation of protrusions by Listeria involves stimulation of host processes apart from actin polymerization. Here we demonstrate that efficient protrusion formation in polarized epithelial cells involves bacterial subversion of host exocytosis. Confocal microscopy imaging indicated that exocytosis is up-regulated in protrusions of Listeria in a manner that depends on the host exocyst complex. Depletion of components of the exocyst complex by RNA interference inhibited the formation of Listeria protrusions and subsequent cell-to-cell spread of bacteria. Additional genetic studies indicated important roles for the exocyst regulators Rab8 and Rab11 in bacterial protrusion formation and spread. The secreted Listeria virulence factor InlC associated with the exocyst component Exo70 and mediated the recruitment of Exo70 to bacterial protrusions. Depletion of exocyst proteins reduced the length of Listeria protrusions, suggesting that the exocyst complex promotes protrusion elongation. Collectively, these results demonstrate that Listeria exploits host exocytosis to stimulate intercellular spread of bacteria.

Role of internalin proteins in the pathogenesis of Listeria monocytogenes.

Listeria monocytogenes is a food-borne bacterium that causes gastroenteritis, meningitis, or abortion. L. monocytogenes induces its internalization (entry) into human cells and either spreads laterally in tissues or transcytoses to traverse anatomical barriers. In this review, we discuss mechanisms by which five structurally related proteins of the "internalin" family of L. monocytogenes (InlA, InlB, InlC, InlF, and InlP) interact with distinct host receptors to promote infection of human cells and/or crossing of the intestinal, blood-brain, or placental barriers. We focus on recent results demonstrating that the internalin proteins InlA, InlB, and InlC exploit exocytic pathways to stimulate transcytosis, entry, or cell-to-cell spread, respectively. We also discuss evidence that InlA-mediated transcytosis contributes to traversal of the intestinal barrier, whereas InlF promotes entry into endothelial cells to breach the blood-brain barrier. InlB also facilitates the crossing of the blood-brain barrier, but does so by extending the longevity of infected monocytes that may subsequently act as a "Trojan horse" to transfer bacteria to the brain. InlA, InlB, and InlP each contribute to fetoplacental infection by targeting syncytiotrophoblast or cytotrophoblast layers of the placenta. This work highlights the diverse functions of internalins and the complex mechanisms by which these structurally related proteins contribute to disease.

Overexpression of a newly identified d-amino acid transaminase in Mycobacterium smegmatis complements glutamate racemase deletion.

Glutamate racemase (MurI) has been proposed as a target for anti-tuberculosis drug development based on the inability of ΔmurI mutants of Mycobacterium smegmatis to grow in the absence of d-glutamate. In this communication, we identify ΔmurI suppressor mutants that are detected during prolonged incubation. Whole genome sequencing of these ΔmurI suppressor mutants identified the presence of a SNP, located in the promoter region of MSMEG_5795. RT-qPCR and transcriptional fusion analyses revealed that the ΔmurI suppressor mutant overexpressed MSMEG_5795 14-fold compared to the isogenic wild-type. MSMEG_5795, which is annotated as 4-amino-4-deoxychorismate lyase (ADCL) but which also has homology to d-amino acid transaminase (d-AAT), was expressed, purified and found to have d-AAT activity and to be capable of producing d-glutamate from d-alanine. Consistent with its d-amino acid transaminase function, overexpressed MSMEG_5795 is able to complement both ΔmurI deletion mutants and alanine racemase (Δalr) deletion mutants, thus confirming a multifunctional role for this enzyme in M. smegmatis.

Investigation of the essentiality of glutamate racemase in Mycobacterium smegmatis.

The mycobacterial cell wall frequently has been used as a target for drug development, and d-glutamate, synthesized by glutamate racemase (MurI), is an important component of peptidoglycan. While the essentiality of the murI gene has been shown in several bacterial species, including Escherichia coli, Bacillus anthracis, and Streptococcus pneumoniae, studies in mycobacteria have not yet provided definitive results. This study aimed to determine whether murI is indeed essential and can serve as a possible target for structure-aided drug design. We have achieved this goal by creating a ΔmurI strain of Mycobacterium smegmatis, a close relative of Mycobacterium tuberculosis. The deletion of the murI gene in M. smegmatis could be achieved only in minimal medium supplemented with D-glutamate, demonstrating that MurI is essential for growth and that glutamate racemase is the only source of D-glutamate for peptidoglycan synthesis in M. smegmatis.

Molecular Mechanisms of Intercellular Dissemination of Bacterial Pathogens.

Several intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexerni, and Rickettsia spp. use an actin-based motility process to spread in mammalian cell monolayers. Cell-to-cell spread is mediated by protrusive structures that contain bacteria encased in the host cell plasma membrane. These protrusions, which form in infected host cells, are internalized by neighboring cells. In this review, we summarize key findings on cell-to-cell spread, focusing on recent work on mechanisms of protrusion formation and internalization. We also discuss the dynamic behavior of bacterial populations during spread, and highlight recent findings showing that intercellular spread by an extracellular bacterial pathogen.

Detection of D-glutamate production from the dual Function enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis.

D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis was identified as having D-amino acid transaminase (D-AAT) activity, although it has primarily been annotated as 4-amino-4-deoxychorismate lyase (ADCL). To unequivocally demonstrate D-AAT activity from MSMEG_5795 protein two coupled enzyme assays were performed in series. First, D-alanine and α-ketoglutarate were converted to D-glutamate and pyruvate by MSMEG_5795 using the D-AAT assay. Next, the products of this reaction, following removal of all protein, were used as input into an assay for glutamate racemase in which D-glutamate is converted to L-glutamate by glutamate racemase (Gallo and Knowles, 1993; Poen et al., 2016 ). As the only source of D-glutamate in this assay would be from the reaction of D-alanine with MSMEG_5795, positive results from this assay would confirm the D-AAT activity of MSMEG_5795 and of any enzyme tested in this manner.

Biomarkers for diagnosis of neonatal infections: A systematic analysis of their potential as a point-of-care diagnostics.

BACKGROUND: Neonatal infections annually claim lives of 1.4 million neonates worldwide. Until now, there is no ideal diagnostic test for detecting sepsis and thus management of possible sepsis cases often depends on clinical algorithm leading to empirical treatment. This often results in unnecessary antibiotic use, which may lead to emergence of antibiotic resistance. Biomarkers have shown great promise in diagnosis of sepsis and guiding appropriate treatment of neonates. In this study, we conducted a literature review of existing biomarkers to analyze their status for use as a point-of-care diagnostic in developing countries. METHODS: PubMed and EMBASE database were searched with keywords, 'infections', 'neonates', and 'biomarkers' to retrieve potentially relevant papers from the period 1980 to 2010. Leading hospitals and manufacturers were communicated to inquire about the cost, laboratory requirements and current standing of biomarkers in clinical use. RESULTS: The search returned 6407 papers on biomarkers; 65 were selected after applying inclusion and exclusion criteria. Among the studies, C-reactive protein (CRP), procalcitonin (PCT) and interleukin 6 (IL-6) were the most widely studied biomarkers and were considered to be most promising for diagnosing neonatal infections. About 90% of the studies were from developed countries; more than 50% were from Europe. CONCLUSIONS: Extensive work is being performed to find the diagnostic and prognostic value of biomarkers. However, the methodologies and study design are highly variable. Despite numerous research papers on biomarkers, their use in clinical setting is limited to CRP. The methods for detection of biomarkers are far too advanced to be used at the community level where most of the babies are dying. It is important that a harmonized multi-site study is initiated to find a battery of biomarkers for diagnosis of neonatal infections.