Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system

Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI-1 and SPI-2 to transfer effector proteins into host cells. A major function of the SPI-1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI-2 is to facilitate the replication of intracellular bacteria within membrane-bound Salmonella-containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI-2-dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase-dependent killing, the induction of a delayed apoptosis-like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI-2-dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments.     

FliZ regulates expression of the Salmonella pathogenicity island 1 invasion locus by controlling HilD protein activity in Salmonella enterica serovar typhimurium

A prerequisite for Salmonella enterica to cause both intestinal and systemic disease is the direct injection of effector proteins into host intestinal epithelial cells via a type three secretion system (T3SS); the T3SS genes are carried on Salmonella pathogenicity island 1 (SPI1). These effector proteins induce inflammatory diarrhea and bacterial invasion. Expression of the SPI1 T3SS is tightly regulated in response to environmental signals through a variety of global regulatory systems. We have previously shown that three AraC-like regulators, HilD, HilC, and RtsA, act in a complex feed-forward regulatory loop to control the expression of the hilA gene, which encodes the direct regulator of the SPI1 structural genes. In this work, we characterize a major positive regulator of this system, the flagellar protein FliZ. Through genetic and biochemical analyses, we show that FliZ posttranslationally controls HilD to positively regulate hilA expression. This mechanism is independent of other flagellar components and is not mediated through the negative regulator HilE or through FliZ-mediated RpoS regulation. We demonstrate that FliZ controls HilD protein activity and not stability. FliZ regulates HilD in the absence of Lon protease, previously shown to degrade HilD. Indeed, it appears that FliZ, rather than HilD, is the most relevant target of Lon as it relates to SPI1 expression. Mutants lacking FliZ are significantly attenuated in their ability to colonize the intestine but are unaffected during systemic infection. The intestinal attenuation is partially dependent on SPI1, but FliZ has additional pleiotropic effects.     

PipB2 is a substrate of the Salmonella pathogenicity island 1-encoded type III secretion system

Salmonella harbors two type III secretion systems, T3SS1 and T3SS2, encoded on the pathogenicity islands SPI1 and SPI2, respectively. Several effector proteins are secreted through these systems into the eukaryotic host cells. PipB2 is a T3SS2 effector that contributes to the modulation of kinesin-1 motor complex activity. Here, we show that PipB2 is also a substrate of T3SS1. This result was obtained infecting human epithelial HeLa cells for 2 h and was confirmed in murine RAW264.7 macrophages, and rat NRK fibroblasts. Analysis at different time points after infection revealed that translocation of PipB2 is T3SS1-dependent in epithelial cells throughout the infection. In contrast, translocation into macrophages is T3SS1-dependent during invasion but T3SS2-dependent at later time points. The N-terminal 10 amino acid residues contain the signal necessary for translocation through both systems. These results confirm the functional overlap between these virulence-related secretion systems and suggest a new role for the effector PipB2.     

SsaM and SpiC interact and regulate secretion of Salmonella pathogenicity island 2 type III secretion system effectors and translocators

The type III secretion system (TTSS) encoded by Salmonella Pathogenicity Island 2 (SPI-2) is required for systemic infection and intracellular replication of Salmonella enterica serovar Typhimurium. The SPI-2 TTSS is activated after internalization of bacteria by host cells, and translocates effector proteins into and across the vacuolar membrane, where they interfere with several host cell functions. Here, we investigated the function of SsaM, a small protein encoded within SPI-2. An ssaM deletion mutant had virulence and intracellular replication defects comparable to those of a SPI-2 TTSS null mutant. Although the ssaM mutant was able to secrete the effector protein SseJ in vitro, it failed to translocate SseJ into host cells, and to secrete the translocon proteins SseB, SseC and SseD in vitro. This phenotype is similar to that of a strain carrying a mutation in the SPI-2 gene spiC, whose product is reported to be an effector involved in trafficking of the Salmonella vacuole in macrophages. Both ssaM and spiC mutants were found to oversecrete the SPI-2 effector proteins SseJ and PipB in vitro. Fractionation assays and immunofluorescence microscopy were used to investigate the localization of SsaM and SpiC in macrophages. No evidence for translocation of these proteins was obtained. The similar phenotypes of the ssaM and spiC mutants suggested that they might be involved in the same function. Pull-down and co-immune precipitation experiments showed that SpiC and SsaM interact within the bacterial cell. We propose that a complex involving SsaM and SpiC distinguishes between translocators and effector proteins, and controls their ordered secretion through the SPI-2 TTSS.     

Effect of the Salmonella pathogenicity island 2 type III secretion system on Salmonella survival in activated chicken macrophage-like HD11 cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment, as E. coli is effectively eliminated.     

Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane

Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.     

Acidic pH is required for the functional assembly of the type III secretion system encoded by Salmonella pathogenicity island 2

Salmonella enterica employs two type III secretion systems (T3SS) for interactions with host cells during pathogenesis. The T3SS encoded by Salmonella pathogenicity island 2 (SPI2) is required for the intracellular replication of Salmonella and the survival inside phagocytes. During growth in vitro, acidic pH is a signal that promotes secretion of proteins by this T3SS. We analyzed protein levels and subcellular localization of various T3SS subunits under in vitro conditions at acidic or neutral pH, inducing or ablating secretion, respectively. Growth at acidic pH resulted in higher levels of SsaC, a protein forming the outer membrane secretin, without increasing expression of the operon containing ssaC. Acidic pH also induced oligomerization of SsaC subunits, a prerequisite for a functional secretin pore. It has previously been described that environmental stimuli resembling the intraphagosomal habitat of Salmonella control the expression of SPI2 genes. Here we propose that such stimuli also modulate the assembly of a functional T3SS that is capable of translocation of effector proteins into the host cell.     

SseBCD proteins are secreted by the type III secretion system of Salmonella pathogenicity island 2 and function as a translocon

The type III secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is required for systemic infections and intracellular accumulation of Salmonella enterica. This system is induced by intracellular Salmonella and subsequently transfers effector proteins into the host cell. Growth conditions either inducing expression of the type III secretion system or the secretion of substrate proteins were defined. Here we report the identification of a set of substrate proteins consisting of SseB, SseC, and SseD that are secreted by the SPI2 system in vitro. Secretion was observed if bacterial cells were exposed to acidic pH after growth in minimal medium with limitation of Mg(2+) or phosphate. SseB, -C, and -D were isolated in a fraction detached from the bacterial cell surface by mechanical shearing, indicating that these proteins are predominantly assembled into complexes on the bacterial cell surface. The three proteins were required for the translocation of SPI2 effector proteins SspH1 and SspH2 into infected host cells. Thus, SseB, SseC, and SseD function as the translocon for effector proteins by intracellular Salmonella.     

Salmonella pathogenicity islands encoding type III secretion systems.

Salmonella enterica harbours two Salmonella pathogenicity islands (SPIs) each encoding a type III secretion system for virulence proteins. SPI1 is required for invasion, while systemic infections and intracellular accumulation of Salmonella are dependent on SPI2 function. This review will describe and compare the genetic organisation, evolution, regulation and molecular functions of SPI1 and SPI2.     

Cloning and transfer of the Salmonella pathogenicity island 2 type III secretion system for studies of a range of gram-negative genera

The engineering of bacterial strains with specific phenotypes frequently requires the use of blocks or "cassettes" of genes that act together to perform a desired function. The potential benefits of utilizing type III secretion systems in this regard are becoming increasingly realized since these systems can be used to direct interactions with host cells for beneficial purposes such as vaccine development, anticancer therapies, and targeted protein delivery. However, convenient methods to clone and transfer type III secretion systems for studies of a range of different types of bacteria are lacking. In addition to functional applications, such methods would also reveal important information about the evolution of a given type III secretion system, such as its ability to be expressed and functional outside of the strain of origin. We describe here the cloning of the Salmonella enterica serovar Typhimurium pathogenicity island 2 (SPI-2) type III secretion system onto a vector that can be easily transferred to a range of gram-negative bacterial genera. We found that expression of the cloned SPI-2 system in different Gammaproteobacteria and Alphaproteobacteria (as monitored by SseB protein levels) is dependent on the bacterial strain and growth medium. We also demonstrate that the cloned system is functional for secretion, can direct interactions with macrophages, and can be used as a novel tool to analyze the predicted interaction of SseB with host cells. This work provides a foundation for future applications where the cloned SPI-2 region (or other cloned type III systems) can provide a desired function to an engineered gram-negative strain.