ORCID Profile
0000-0002-0529-9406
Current Organisations
Washington State University
,
University of Washington
,
Genentech Inc
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Publisher: Springer New York
Date: 05-11-2016
DOI: 10.1007/978-1-4939-6581-6_19
Abstract: Establishment of an intracellular niche within mammalian cells is key to the pathogenesis of the gastrointestinal bacterium, Salmonella enterica serovar Typhimurium (S. Typhimurium). Here we will describe how to study the internalization of S. Typhimurium into human epithelial cells using the gentamicin protection assay. The assay takes advantage of the relatively poor penetration of gentamicin into mammalian cells internalized bacteria are effectively protected from its antibacterial actions. A second assay, the chloroquine (CHQ) resistance assay, can be used to determine the proportion of internalized bacteria that have lysed or damaged their Salmonella-containing vacuole and are therefore residing within the cytosol. Its application to the quantification of cytosolic S. Typhimurium in epithelial cells will also be presented. Together, these protocols provide an inexpensive, rapid and sensitive quantitative measure of bacterial internalization and vacuole lysis by S. Typhimurium.
Publisher: American Society for Microbiology
Date: 29-12-2017
Abstract: The Salmonella invasion-associated type III secretion system (T3SS1) is an essential virulence factor required for entry into nonphagocytic cells and consequent uptake into a Salmonella -containing vacuole (SCV). While Salmonella is typically regarded as a vacuolar pathogen, a subset of bacteria escape from the SCV in epithelial cells and eventually hyperreplicate in the cytosol. T3SS1 is downregulated following bacterial entry into mammalian cells, but cytosolic Salmonella cells are T3SS1 induced, suggesting prolonged or resurgent activity of T3SS1 in this population. In order to investigate the postinternalization contributions of T3SS1 to the Salmonella infectious cycle in epithelial cells, we bypassed its requirement for bacterial entry by tagging the T3SS1-energizing ATPase InvC at the C terminus with peptides that are recognized by bacterial tail-specific proteases. This caused a dramatic increase in InvC turnover which rendered even assembled injectisomes inactive. Bacterial strains conditionally expressing these unstable InvC variants were proficient for invasion but underwent rapid and sustained intracellular inactivation of T3SS1 activity when InvC expression ceased. This allowed us to directly implicate T3SS1 activity in cytosolic colonization and bacterial egress. We subsequently identified two T3SS1-delivered effectors, SopB and SipA, that are required for efficient colonization of the epithelial cell cytosol. Overall, our findings support a multifaceted, postinvasion role for T3SS1 and its effectors in defining the cytosolic population of intracellular Salmonella . IMPORTANCE A needle-like apparatus, the type III secretion system (T3SS) injectisome, is absolutely required for Salmonella enterica to enter epithelial cells this requirement has h ered the analysis of its postentry contributions. To identify T3SS1-dependent intracellular activities, in this study we overcame this limitation by developing a conditional inactivation in the T3SS whereby T3SS activity is chemically induced during culture in liquid broth, permitting bacterial entry into epithelial cells, but is quickly and perpetually inactivated in the absence of inducer. In this sense, the mutant acts like wild-type bacteria when extracellular and as a T3SS mutant once it enters a host cell. This “conditional” mutant allowed us to directly link activity of this T3SS with nascent vacuole lysis, cytosolic proliferation, and cellular egress, demonstrating that the invasion-associated T3SS also contributes to essential intracellular stages of the S. enterica infectious cycle.
Publisher: Proceedings of the National Academy of Sciences
Date: 12-04-2016
Abstract: Bacterial pathogens use a variety of strategies to evade host cell innate immune responses. For some, this includes the establishment of an intracellular replicative niche. Although many intracellular pathogens remodel phagosomes to support bacterial replication, others lyse their internalization vacuole to reside within the host cell cytosol. Little is currently known regarding how bacteria, particularly Gram-negative pathogens, mediate phagosomal escape. Using complementary reductionist and functional interchangeability experimental approaches, we demonstrate that the type III secretion system machinery itself directly modulates the extent to which bacteria escape from phagosomes. Given the high prevalence of type III secretion systems among intracellular bacterial pathogens, these studies have identified a potential means by which the intracellular niche of Gram-negative bacteria is defined.
Publisher: Wiley
Date: 30-01-2017
DOI: 10.1111/MMI.13602
Abstract: Type III Secretion Systems (T3SSs) are structurally conserved nanomachines that span the inner and outer bacterial membranes, and via a protruding needle complex contact host cell membranes and deliver type III effector proteins. T3SS are phylogenetically ided into several families based on structural basal body components. Here we have studied the evolutionary and functional conservation of four T3SS proteins from the Inv/Mxi-Spa family: a cytosolic chaperone, two hydrophobic translocators that form a plasma membrane-integral pore, and the hydrophilic 'tip complex' translocator that connects the T3SS needle to the translocon pore. Salmonella enterica serovar Typhimurium (S. Typhimurium), a common cause of food-borne gastroenteritis, possesses two T3SSs, one belonging to the Inv/Mxi-Spa family. We used invasion-deficient S. Typhimurium mutants as surrogates for expression of translocator orthologs identified from an extensive phylogenetic analysis, and type III effector translocation and host cell invasion as a readout for complementation efficiency, and identified several Inv/Mxi-Spa orthologs that can functionally substitute for the S. Typhimurium chaperone and translocator proteins. Functional complementation correlates with amino acid sequence identity between orthologs, but varies considerably between the four proteins. This is the first in-depth survey of the functional interchangeability of Inv/Mxi-Spa T3SS proteins acting directly at the host-pathogen interface.
Location: United States of America
No related grants have been discovered for Jessica Klein.