Flagella-mediated cytosolic motility of Salmonella enterica Paratyphi A aids in evasion of xenophagy but does not impact egress from host cells.

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal serovars like Paratyphi A (SPA) are human restricted and cause severe systemic diseases, while many serovars like Typhimurium (STM) have a broad host range, and usually lead to self-limiting gastroenteritis. There are key differences between typhoidal and non-typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Transcriptomes and phenotypes in epithelial cells revealed induction of motility, flagella and chemotaxis genes for SPA but not STM. SPA exhibited cytosolic motility mediated by flagella. In this study, we applied single-cell microscopy to analyze triggers and cellular consequences of cytosolic motility. Live-cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at invasion sites led to increased membrane damage in nascent Salmonella-containing vacuole, and subsequent cytosolic release. After release into the cytosol, motile bacteria showed the same velocity as under culture conditions in media. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Prior work showed that SPA does not use flagella-mediated motility for cell exit via the intercellular spread. However, cytosolic motile SPA was invasion-primed if released from host cells. Our results reveal flagella-mediated cytosolic motility as a possible xenophagy evasion mechanism that could drive disease progression and contributes to the dissemination of systemic infection.

Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes.

The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair.

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

Intracellular Salmonella Paratyphi A is motile and differs in the expression of flagella-chemotaxis, SPI-1 and carbon utilization pathways in comparison to intracellular S. Typhimurium.

Although Salmonella Typhimurium (STM) and Salmonella Paratyphi A (SPA) belong to the same phylogenetic species, share large portions of their genome and express many common virulence factors, they differ vastly in their host specificity, the immune response they elicit, and the clinical manifestations they cause. In this work, we compared their intracellular transcriptomic architecture and cellular phenotypes during human epithelial cell infection. While transcription induction of many metal transport systems, purines, biotin, PhoPQ and SPI-2 regulons was similar in both intracellular SPA and STM, we identified 234 differentially expressed genes that showed distinct expression patterns in intracellular SPA vs. STM. Surprisingly, clear expression differences were found in SPI-1, motility and chemotaxis, and carbon (mainly citrate, galactonate and ethanolamine) utilization pathways, indicating that these pathways are regulated differently during their intracellular phase. Concurring, on the cellular level, we show that while the majority of STM are non-motile and reside within Salmonella-Containing Vacuoles (SCV), a significant proportion of intracellular SPA cells are motile and compartmentalized in the cytosol. Moreover, we found that the elevated expression of SPI-1 and motility genes by intracellular SPA results in increased invasiveness of SPA, following exit from host cells. These findings demonstrate unexpected flagellum-dependent intracellular motility of a typhoidal Salmonella serovar and intriguing differences in intracellular localization between typhoidal and non-typhoidal salmonellae. We propose that these differences facilitate new cycles of host cell infection by SPA and may contribute to the ability of SPA to disseminate beyond the intestinal lamina propria of the human host during enteric fever.

Manipulation of Host Cell Organelles by Intracellular Pathogens.

Pathogenic intracellular bacteria, parasites and viruses have evolved sophisticated mechanisms to manipulate mammalian host cells to serve as niches for persistence and proliferation. The intracellular lifestyles of pathogens involve the manipulation of membrane-bound organellar compartments of host cells. In this review, we described how normal structural organization and cellular functions of endosomes, endoplasmic reticulum, Golgi apparatus, mitochondria, or lipid droplets are targeted by microbial virulence mechanisms. We focus on the specific interactions of Salmonella, Legionella pneumophila, Rickettsia rickettsii, Chlamydia spp. and Mycobacterium tuberculosis representing intracellular bacterial pathogens, and of Plasmodium spp. and Toxoplasma gondii representing intracellular parasites. The replication strategies of various viruses, i.e., Influenza A virus, Poliovirus, Brome mosaic virus, Epstein-Barr Virus, Hepatitis C virus, severe acute respiratory syndrome virus (SARS), Dengue virus, Zika virus, and others are presented with focus on the specific manipulation of the organelle compartments. We compare the specific features of intracellular lifestyle and replication cycles, and highlight the communalities in mechanisms of manipulation deployed.

Single cell analyses reveal distinct adaptation of typhoidal and non-typhoidal Salmonella enterica serovars to intracellular lifestyle.

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Human-restricted typhoidal S. enterica serovars Typhi (STY) or Paratyphi A (SPA) cause severe typhoid or paratyphoid fever, while many S. enterica serovar Typhimurium (STM) strains have a broad host range and in human hosts usually lead to a self-limiting gastroenteritis. Due to restriction of STY and SPA to primate hosts, experimental systems for studying the pathogenesis of typhoid and paratyphoid fever are limited. Therefore, STM infection of susceptible mice is commonly considered as model system for studying these diseases. The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2-T3SS) is a key factor for intracellular survival of Salmonella. Inside host cells, the pathogen resides within the Salmonella-containing vacuole (SCV) and induces tubular structures extending from the SCV, termed Salmonella-induced filaments (SIF). This study applies single cell analyses approaches, which are flow cytometry of Salmonella harboring dual fluorescent protein reporters, effector translocation, and correlative light and electron microscopy to investigate the fate and activities of intracellular STY and SPA. The SPI2-T3SS of STY and SPA is functional in translocation of effector proteins, SCV and SIF formation. However, only a low proportion of intracellular STY and SPA are actively deploying SPI2-T3SS and STY and SPA exhibited a rapid decline of protein biosynthesis upon experimental induction. A role of SPI2-T3SS for proliferation of STY and SPA in epithelial cells was observed, but not for survival or proliferation in phagocytic host cells. Our results indicate that reduced intracellular activities are factors of the stealth strategy of STY and SPA and facilitate systemic spread and persistence of the typhoidal Salmonella.

Pathoadaptation of the passerine-associated Salmonella enterica serovar Typhimurium lineage to the avian host.

Salmonella enterica is a diverse bacterial pathogen and a primary cause of human and animal infections. While many S. enterica serovars present a broad host-specificity, several specialized pathotypes have been adapted to colonize and cause disease in one or limited numbers of host species. The underlying mechanisms defining Salmonella host-specificity are far from understood. Here, we present genetic analysis, phenotypic characterization and virulence profiling of a monophasic S. enterica serovar Typhimurium strain that was isolated from several wild sparrows in Israel. Whole genome sequencing and complete assembly of its genome demonstrate a unique genetic signature that includes the integration of the BTP1 prophage, loss of the virulence plasmid, pSLT and pseudogene accumulation in multiple T3SS-2 effectors (sseJ, steC, gogB, sseK2, and sseK3), catalase (katE), tetrathionate respiration (ttrB) and several adhesion/ colonization factors (lpfD, fimH, bigA, ratB, siiC and siiE) encoded genes. Correspondingly, this strain demonstrates impaired biofilm formation, intolerance to oxidative stress and compromised intracellular replication within non-phagocytic host cells. Moreover, while this strain showed attenuated pathogenicity in the mouse, it was highly virulent and caused an inflammatory disease in an avian host. Overall, our findings demonstrate a unique phenotypic profile and genetic makeup of an overlooked S. Typhimurium sparrow-associated lineage and present distinct genetic signatures that are likely to contribute to its pathoadaptation to passerine birds.