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.     

沙门菌毒力岛引发持续性感染机制的研究进展

沙门菌是一种重要的人兽共患食源性病原菌。其感染宿主后可以凭借独特的免疫逃逸机制逃避宿主免疫系统的清除,潜伏在宿主体内1年至终身不等,从而建立持续性感染。沙门菌持续性感染与毒力岛密切相关,尤其是沙门菌毒力岛（Salmonella pathogenicity islands,SPIs） SPI-1和SPI-2。SPI-1效应蛋白SipB和SipC等以不同的途径影响细菌入侵,诱导细胞自噬或者凋亡;而SPI-2效应蛋白SseI和SseL等可以通过调控不同的信号通路协助沙门菌的胞内存活,为沙门菌持续性感染的发生和发展提供条件。本文主要阐述SipB和SseI等毒力岛效应蛋白在沙门菌持续性感染过程中的作用,同时总结了SPI-6、SPI-7和SPI-19等毒力岛的作用,以期为研究沙门菌持续性感染提供新思路。     

Human NAIP/NLRC4 and NLRP3 inflammasomes detect Salmonella type III secretion system activities to restrict intracellular bacterial replication

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that uses two distinct type III secretion systems (T3SSs), termed Salmonella pathogenicity island (SPI)-1 and SPI-2, to deliver virulence factors into the host cell. The SPI-1 T3SS enables Salmonella to invade host cells, while the SPI-2 T3SS facilitates Salmonella’s intracellular survival. In mice, a family of cytosolic immune sensors, including NAIP1, NAIP2, and NAIP5/6, recognizes the SPI-1 T3SS needle, inner rod, and flagellin proteins, respectively. Ligand recognition triggers assembly of the NAIP/NLRC4 inflammasome, which mediates caspase-1 activation, IL-1 family cytokine secretion, and pyroptosis of infected cells. In contrast to mice, humans encode a single NAIP that broadly recognizes all three ligands. The role of NAIP/NLRC4 or other inflammasomes during Salmonella infection of human macrophages is unclear. We find that although the NAIP/NLRC4 inflammasome is essential for detecting T3SS ligands in human macrophages, it is partially required for responses to infection, as Salmonella also activated the NLRP3 and CASP4/5 inflammasomes. Importantly, we demonstrate that combinatorial NAIP/NLRC4 and NLRP3 inflammasome activation restricts Salmonella replication in human macrophages. In contrast to SPI-1, the SPI-2 T3SS inner rod is not sensed by human or murine NAIPs, which is thought to allow Salmonella to evade host recognition and replicate intracellularly. Intriguingly, we find that human NAIP detects the SPI-2 T3SS needle protein. Critically, in the absence of both flagellin and the SPI-1 T3SS, the NAIP/NLRC4 inflammasome still controlled intracellular Salmonella burden. These findings reveal that recognition of Salmonella SPI-1 and SPI-2 T3SSs and engagement of both the NAIP/NLRC4 and NLRP3 inflammasomes control Salmonella infection in human macrophages.     

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

Contribution of the Type VI Secretion System Encoded in SPI-19 to Chicken Colonization by Salmonella enterica Serotypes Gallinarum and Enteritidis

Salmonella Gallinarum is a pathogen with a host range specific to poultry, while Salmonella Enteritidis is a broad host range pathogen that colonizes poultry sub-clinically but is a leading cause of gastrointestinal salmonellosis in humans and many other species. Despite recent advances in our understanding of the complex interplay between Salmonella and their hosts, the molecular basis of host range restriction and unique pathobiology of Gallinarum remain largely unknown. Type VI Secretion System (T6SS) represents a new paradigm of protein secretion that is critical for the pathogenesis of many Gram-negative bacteria. We recently identified a putative T6SS in the Salmonella Pathogenicity Island 19 (SPI-19) of Gallinarum. In Enteritidis, SPI-19 is a degenerate element that has lost most of the T6SS functions encoded in the island. In this work, we studied the contribution of SPI-19 to the colonization of Salmonella Gallinarum strain 287/91 in chickens. Non-polar deletion mutants of SPI-19 and the clpV gene, an essential T6SS component, colonized the ileum, ceca, liver and spleen of White Leghorn chicks poorly compared to the wild-type strain after oral inoculation. Return of SPI-19 to the ΔSPI-19 mutant, using VEX-Capture, complemented this colonization defect. In contrast, transfer of SPI-19 from Gallinarum to Enteritidis resulted in transient increase in the colonization of the ileum, liver and spleen at day 1 post-infection, but at days 3 and 5 post-infection a strong colonization defect of the gut and internal organs of the experimentally infected chickens was observed. Our data indicate that SPI-19 and the T6SS encoded in this region contribute to the colonization of the gastrointestinal tract and internal organs of chickens by Salmonella Gallinarum and suggest that degradation of SPI-19 T6SS in Salmonella Enteritidis conferred an advantage in colonization of the avian host.     

The Salmonella Deubiquitinase SseL Inhibits Selective Autophagy of Cytosolic Aggregates

Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of Salmonella enterica occurs in a membranous compartment, the Salmonella-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the Salmonella pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular Salmonella replication. Our data therefore show that there is a host selective autophagy response to intracellular Salmonella infection, which is counteracted by the deubiquitinase SseL.     

HLA-B27 modulates intracellular growth of Salmonella pathogenicity island 2 mutants and production of cytokines in infected monocytic U937 cells

Background

Salmonella enterica serovar Enteritidis PT4 KS8822/88 replicates rapidly in HLA-B27-transfected human monocytic U937 cells. In this process, Salmonella pathogenicity island 2 (SPI-2) genes play a crucial role. Our previous study indicated that 118 Salmonella genes, including 8 SPI-2 genes were affected by HLA-B27 antigen during Salmonella infection of U937 cells.

Methods/Principal Findings

To further investigate Salmonella replication in HLA-B27-positive U937 monocytic cells, two SPI-2 genes, ssaS and sscA up-regulated most during Salmonella infection of HLA-B27-transfected U937 cells, were mutated by using one-step gene disruption method. Intracellular survival and replication of the mutants in the U937 cells was compared to that of the wild type strain. Surprisingly, the two mutated strains replicated significantly more than the wild type bacteria in HLA-B27-transfected cells. Secretion of tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10) was significantly induced during the infection of HLA-B27-transfected U937 cells with the mutants. The results indicated that the certain SPI-2 genes in wild type bacteria suppress Salmonella intracellular growth and production of cytokines in infected HLA-B27-transfected cells. HLA-B27-associated modulation of Salmonella SPI-2 genes and cytokine production may have importance in the persistent infection of the bacteria and the pathogenesis of reactive arthritis.

Conclusions

The study provides evidence that certain virulence factors of pathogens can reduce the intracellular growth in the host cells. We suggest that the limiting intracellular growth might be a strategy for persistence of bacteria in host cells, keeping a balance between pathogenic growth and pathogenesis.     

The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium: dual regulation of flagellar and SPI-2 virulence genes

The Rcs phosphorelay is a multicomponent signaling system that positively regulates colanic acid synthesis and negatively regulates motility and virulence. We have exploited a spontaneously isolated mutant, IgaA(T191P), that is nearly maximally activated for the Rcs system to identify a vast set of genes that respond to the stimulation, and we report new regulatory properties of this signaling system in Salmonella enterica serovar Typhimurium. Microarray data show that the Rcs system normally functions as a positive regulator of SPI-2 and other genes important for the growth of Salmonella in macrophages, although when highly activated the system completely represses the SPI-1/SPI-2 virulence, flagellar, and fimbrial biogenesis pathways. The auxiliary protein RcsA, which works with RcsB to positively regulate colanic acid and other target genes, not only stimulates but also antagonizes the positive regulation of many genes in the igaA mutant. We show that RcsB represses motility through the RcsB box in the promoter region of the master operon flhDC and that RcsA is not required for this regulation. Curiously, RcsB selectively stimulates expression of the flagellar type 3 secretion genes fliPQR; an RcsAB box located downstream of fliR influences this regulation. We show that excess colanic acid impairs swimming and inhibits swarming motility, consistent with the inverse regulation of the two pathways by the Rcs system.     

Typhoidal Salmonella: Distinctive virulence factors and pathogenesis

Although nontyphoidal Salmonella (NTS; including Salmonella Typhimurium) mainly cause gastroenteritis, typhoidal serovars (Salmonella Typhi and Salmonella Paratyphi A) cause typhoid fever, the treatment of which is threatened by increasing drug resistance. Our understanding of S. Typhi infection in human remains poorly understood, likely due to the host restriction of typhoidal strains and the subsequent popularity of the S. Typhimurium mouse typhoid model. However, translating findings with S. Typhimurium across to S. Typhi has some limitations. Notably, S. Typhi has specific virulence factors, including typhoid toxin and Vi antigen, involved in symptom development and immune evasion, respectively. In addition to unique virulence factors, both typhoidal and NTS rely on two pathogenicity‐island encoded type III secretion systems (T3SS), the SPI‐1 and SPI‐2 T3SS, for invasion and intracellular replication. Marked differences have been observed in terms of T3SS regulation in response to bile, oxygen, and fever‐like temperatures. Moreover, approximately half of effectors found in S. Typhimurium are either absent or pseudogenes in S. Typhi, with most of the remaining exhibiting sequence variation. Typhoidal‐specific T3SS effectors have also been described. This review discusses what is known about the pathogenesis of typhoidal Salmonella with emphasis on unique behaviours and key differences when compared with S. Typhimurium.     

Activation of Focal Adhesion Kinase by Salmonella Suppresses Autophagy via an Akt/mTOR Signaling Pathway and Promotes Bacterial Survival in Macrophages

Autophagy has emerged as an important antimicrobial host defense mechanism that not only orchestrates the systemic immune response, but also functions in a cell autonomous manner to directly eliminate invading pathogens. Pathogenic bacteria such as Salmonella have evolved adaptations to protect themselves from autophagic elimination. Here we show that signaling through the non-receptor tyrosine kinase focal adhesion kinase (FAK) is actively manipulated by the Salmonella SPI-2 system in macrophages to promote intracellular survival. In wild-type macrophages, FAK is recruited to the surface of the Salmonella-containing vacuole (SCV), leading to amplified signaling through the Akt-mTOR axis and inhibition of the autophagic response. In FAK-deficient macrophages, Akt/mTOR signaling is attenuated and autophagic capture of intracellular bacteria is enhanced, resulting in reduced bacterial survival. We further demonstrate that enhanced autophagy in FAK^−/− macrophages requires the activity of Atg5 and ULK1 in a process that is distinct from LC3-assisted phagocytosis (LAP). In vivo, selective knockout of FAK in macrophages resulted in more rapid clearance of bacteria from tissues after oral infection with S. typhimurium. Clearance was correlated with reduced infiltration of inflammatory cell types into infected tissues and reduced tissue damage. Together, these data demonstrate that FAK is specifically targeted by S. typhimurium as a novel means of suppressing autophagy in macrophages, thereby enhancing their intracellular survival.     

Attenuated Salmonella Typhimurium Lacking the Pathogenicity Island-2 Type 3 Secretion System Grow to High Bacterial Numbers inside Phagocytes in Mice

Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.     

假定调控蛋白STM14_3514可降低鼠伤寒沙门菌对上皮细胞的侵袭力

【目的】研究鼠伤寒沙门菌致病岛1(SPI-1)内部的假定调控蛋白STM14_3514的功能及其作用机制。【方法】以鼠伤寒沙门菌模式菌株ATCC 14028为亲本株,构建了STM14_3514基因的缺失突变体及互补菌株,通过小鼠实验、细胞侵袭实验、Western blot及实时荧光定量PCR(q RT-PCR)等实验技术,深入研究了STM14_3514基因对鼠伤寒沙门菌致病过程的影响。【结果】STM14_3514突变提高了细菌对小鼠的致病能力,突变体在小鼠肠道、肝和脾中的定殖能力均增强;细胞实验揭示,突变体致病力提升主要由于STM14_3514突变能显著增强细菌对上皮细胞的侵袭力(2倍,P0.05)。q RT-PCR及Western blot分析表明,STM14_3514显著抑制SPI-1内部主要调控因子hil A及侵袭相关基因的表达。此外,STM14_3514对hil A的抑制由Hil C介导。【结论】STM14_3514是鼠伤寒沙门菌SPI-1内部的负调控因子,能通过Hil C抑制hil A及SPI-1其他入侵基因的表达,该基因的生物学意义可能与细菌进入细胞后对SPI-1的负调控相关。