Many Gram-negative bacterial symbionts and pathogens employ a type III secretion system (T3SS) to live in contact with eukaryotic cells. Because T3SSs inject bacterial proteins (effectors) directly into host cells, the switching of secretory substrates between translocators and effectors in response to host cell attachment is a crucial step for the effective delivery of effectors. Here, we show that the protein secretion switch of Vibrio parahaemolyticus T3SS2, which is a main contributor to the enteropathogenicity of a food poisoning bacterium, is regulated by two gatekeeper proteins, VgpA and VgpB. In the absence of these gatekeepers, effector secretion was activated, but translocator secretion was abolished, causing the loss of virulence. We found that the K+ concentration, which is high inside the host cell but low outside, is a key factor for VgpA- and VgpB-mediated secretion switching. Exposure of wild-type bacteria to K+ ions provoked both gatekeeper and effector secretions but reduced the level of secretion of translocators. The secretion protein profile of wild-type bacteria cultured with 0.1 M KCl was similar to that of gatekeeper mutants. Furthermore, depletion of K+ ions in host cells diminished the efficiency of T3SS2 effector translocation. Thus, T3SS2 senses the high intracellular concentration of K+ of the host cell so that T3SS2 effectors can be effectively injected.IMPORTANCE The pathogenesis of many Gram-negative bacterial pathogens arises from a type III secretion system (T3SS), whereby bacterial proteins (effectors) are directly injected into host cells. The injected effectors then modify host cell functions. For effective delivery of effector proteins, bacteria need to both recognize host cell attachment and switch the type of secreted proteins. Here, we identified gatekeeper proteins that play important roles in a T3SS2 secretion switch of Vibrio parahaemolyticus, a causative agent of food-borne gastroenteritis. We also found that K+, which is present in high concentrations inside the host cell but in low concentrations outside, is a key factor for the secretion switch. Thus, V. parahaemolyticus senses the high intracellular K+ concentration, triggering the effective injection of effectors.

Salmonella Typhi is the causative agent of typhoid fever, which is a lifethreatening, systemic disease, with an estimated global disease burden of 21.6 million cases annually, resulting in about 220,000 deaths. Due to the absence of convenient animals models to study S. Typhi and other typhoidal Salmonella serovars, our understanding of typhoid fever pathogenesis is still incomplete.

Typhoid fever caused by Salmonella enterica serovar (S.) Typhi differs in its clinical presentation from gastroenteritis caused by S. Typhimurium and other non-typhoidal Salmonella serovars. The different clinical presentations are attributed in part to the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi, which prevents phagocytes from triggering a respiratory burst by preventing antibody-mediated complement activation. Paradoxically, the Vi antigen is absent from S. Paratyphi A, which causes a disease that is indistinguishable from typhoid fever. Here, we show that evasion of the phagocyte respiratory burst by S. Paratyphi A required very long O antigen chains containing the O2 antigen to inhibit antibody binding. We conclude that the ability to avoid the phagocyte respiratory burst is a property distinguishing typhoidal from non-typhoidal Salmonella serovars that was acquired by S. Typhi and S. Paratyphi A independently through convergent evolution.

Capsular polysaccharides are common virulence factors of extracellular, but not intracellular bacterial pathogens, due to the antiphagocytic properties of these surface structures. It is therefore paradoxical that Salmonella enterica subspecies enterica serovar Typhi, an intracellular pathogen, synthesizes a virulence-associated (Vi) capsule, which exhibits antiphagocytic properties. Here, we show that the Vi capsular polysaccharide has different functions when S. Typhi interacts with distinct subsets of host phagocytes. The Vi capsular polysaccharide allowed S. Typhi to selectively evade phagocytosis by human neutrophils while promoting human macrophage phagocytosis. A screen of C-type lectin receptors identified human DC-SIGN as the receptor involved in macrophage binding and phagocytosis of capsulated S. Typhi. Consistent with the anti-inflammatory activity of DC-SIGN, purified Vi capsular polysaccharide reduced inflammatory responses in macrophages. These data suggest that binding of the human C-type lectin receptor DC-SIGN by the Vi capsular polysaccharide contributes to the pathogenesis of typhoid fever. IMPORTANCE Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever. The recent emergence of S. Typhi strains which are resistant to antibiotic therapy highlights the importance of vaccination in managing typhoid fever. The virulence-associated (Vi) capsular polysaccharide is an effective vaccine against typhoid fever, but the role the capsule plays during pathogenesis remains incompletely understood. Here, we identify the human C-type lectin receptor DC-SIGN as the receptor for the Vi capsular polysaccharide. Binding of capsulated S. Typhi to DC-SIGN resulted in phagocytosis of the pathogen by macrophages and induction of an anti-inflammatory cytokine response. Thus, the interaction of the Vi capsular polysaccharide with human DC-SIGN contributes to the pathogenesis of typhoid fever and should be further investigated in the context of vaccine development.

Intracellular pathogens commonly reside within macrophages to find shelter from humoral defenses, but host cell death can expose them to the extracellular milieu. We find intracellular pathogens solve this dilemma by using virulence factors to generate a complement-dependent find-me signal that initiates uptake by a new phagocyte through efferocytosis. During macrophage death, Salmonella uses a type III secretion system to perforate the membrane of the pathogen-containing vacuole (PCV), thereby triggering complement deposition on bacteria entrapped in pore-induced intracellular traps (PITs). In turn, complement activation signals neutrophil efferocytosis, a process that shelters intracellular bacteria from the respiratory burst. Similarly, Brucella employs its type IV secretion system to perforate the PCV membrane, which induces complement deposition on bacteria entrapped in PITs. Collectively, this work identifies virulence factor-induced perforation of the PCV as a strategy of intracellular pathogens to generate a find-me signal for efferocytosis.

Changes in the microbiota composition are associated with many human diseases, but factors that govern strain abundance remain poorly defined. We show that a commensal Escherichia coli strain and a pathogenic Salmonella enterica serovar Typhimurium isolate both utilize nitrate for intestinal growth, but each accesses this resource in a distinct biogeographical niche. Commensal E. coli utilizes epithelial-derived nitrate, whereas nitrate in the niche occupied by S. Typhimurium is derived from phagocytic infiltrates. Surprisingly, avirulent S. Typhimurium was shown to be unable to utilize epithelial-derived nitrate because its chemotaxis receptors McpB and McpC exclude the pathogen from the niche occupied by E. coli. In contrast, E. coli invades the niche constructed by S. Typhimurium virulence factors and confers colonization resistance by competing for nitrate. Thus, nutrient niches are not defined solely by critical resources, but they can be further subdivided biogeographically within the host into distinct microhabitats, thereby generating new niche opportunities for distinct bacterial species.