MxiE Regulates Intracellular Expression of Factors Secreted by the Shigella flexneri 2a Type III Secretion System

Members of the genus Shigella are gram-negative, facultative intracellular bacteria that are the causative agents of bacillary dysentery in humans. These organisms invade the colonic mucosa, causing severe inflammation, which eventually leads to mucosal destruction and results in blood and mucus in the stools (30). The key aspect of Shigella pathogenesis lies in its ability to invade epithelial cells and spread directly from cell to cell. Epithelial cell invasion occurs by a process similar to phagocytosis and requires major rearrangements of the host cell cytoskeleton (9). Once inside the eukaryotic cell, the bacteria escape from the phagocytic vacuole and are able to multiply in the cytoplasm and spread to adjacent cells via nucleation of host cell actin by the outer membrane protein IcsA (5). The force of the rapid actin polymerization propels the organisms through the cytosol, leading to formation of membrane-bound protrusions, which are then engulfed by neighboring cells. The resulting double membrane-bound vacuole is lysed by bacterial proteins, IpaB and IpaC, allowing the cycle to repeat (51). The ability of shigellae to invade the host epithelium is dependent upon a 31-kb entry region of the 230-kb virulence plasmid (34). The entry region contains two divergently transcribed loci that encode the genes responsible for invasion of eukaryotic cells, intracellular spread, and production of a type III secretion system. One locus is comprised of the mxi and spa operons, which encodes ca. 20 different proteins that together make up the type III secretion apparatus. Type III, or contact-dependent, secretion systems are present in a wide variety of gram-negative plant and animal pathogens and function to secrete protein effectors out of the bacterium and inject them into the cytosol of eukaryotic host cells (reviewed in reference 27). Effector molecules perform a wide variety of functions, some of which include translocation of other effectors into the host cell and interaction with host cell cytoskeletal components to facilitate entry of the bacteria. Although the effector proteins are unique to each bacterial species, the proteins comprising the structural apparatus are highly conserved even in distantly related organisms. Some of the events leading to bacterial uptake, such as cytoskeletal rearrangements, are mediated through secreted or translocated effectors. In Shigella, the entry region of the virulence plasmid also contains a cluster of genes encoding the type III secreted effector molecules (the invasion plasmid antigens, IpaA, IpaB, IpaC, and IpaD), which are required for host cell invasion and escape from the phagosome (37). Furthermore, ipgC, which encodes a molecular chaperone that associates with IpaB and IpaC in the bacterial cytoplasm, is found immediately upstream of the ipa genes in this region. While not secreted, IpgC is required for bacterial entry by stabilizing IpaB and IpaC and preventing their premature association (38). During a natural infection, secretion of presynthesized effectors is triggered by contact with a eukaryotic cell. However, in the laboratory, type III secretion can be artificially induced in the absence of host cell contact by manipulation of the growth medium (e.g., removal of Ca2+ for Yersinia) or by the addition of specific chemicals, such as Congo red for Shigella (3). Expression of the type III apparatus and its effectors is usually regulated by environmental factors, such as temperature, and often regulated at the level of transcription. In Shigella, the AraC-like protein, VirF, is well established as the primary regulator of virulence gene expression. When the bacteria encounter a 37°C environment, VirF activates transcription of both icsA and the secondary regulatory gene virB, thereby initiating the cascade of virulence gene expression, although exactly how VirF responds to temperature is not yet known (45). This thermal activation is not a result of de novo virF transcription since functional VirF protein is present in the bacterial cytosol even at nonpermissive temperatures but rather is due to changes in the amount of supercoiling at the virB promoter which allows VirF to bind and activate its transcription (55). VirB, in turn, activates transcription of the ipa, mxi, and spa operons, which encode the type III secretion system that is essential for invasion (54). A common mechanism of gene regulation in bacteria is via regulatory proteins of the AraC/XylS family. To date, this family contains more than 100 members as identified by sequence homology to the AraC protein of Escherichia coli (19). With few exceptions, AraC homologues are transcriptional activators. The defining characteristic of this family of proteins is a C-terminal 99-amino-acid stretch of homology comprising the DNA-binding domain. Importantly, this region contains two helix-turn-helix motifs, at least one of which is responsible for binding to the major groove of DNA. A separate domain (sensor domain) or additional protein acts to bind effector molecules. Members of the AraC/XylS family regulate a broad variety of bacterial functions, which can be divided into three categories: carbon metabolism, stress response, and virulence. The AraC homologues that regulate virulence gene expression have not been shown to bind effectors directly, although they do respond to environmental signals such as temperature, osmolarity, or calcium concentration by an unidentified mechanism (19). In addition to VirF (mentioned above), the role of several AraC homologues that are involved in bacterial pathogenesis has been the focus of intense research. Many of these proteins function as transcriptional activators of molecules that are secreted through the type III secretion system, their cognate chaperones, and in some cases, type III secretion apparatus structural components as well. The AraC-like InvF protein from Salmonella enterica serotype Typhimurium is required for efficient invasion into cultured epithelial cells and activates the expression of genes whose products are secreted through the type III secretion system (12, 28). InvF is encoded by the first gene of the large inv operon and acts upon the sigDE and sicA-sipBCDA operons (12). SigD and the Sip proteins are secreted through the type III apparatus whereas SigE and SicA function as chaperones for these effectors (13, 26, 29). In contrast to other enteric pathogens, transcription of the type III secretion genes in Salmonella is not regulated by temperature. These organisms respond to other environmental signals, including oxygen concentration, osmolarity, and pH (4). VirF, an AraC homologue of Yersinia enterocolitica, functions as a transcriptional activator of several plasmid-encoded virulence genes: yopE, yopH, and the lcrGVH-yopBD and yscB-yscM operons (31). These Yops are secreted effectors that facilitate formation of channels in the eukaryotic membrane or are translocated into the eukaryotic cell where they are involved in the modulation of cellular signaling (6, 25, 58). LcrG, LcrV, and LcrH each perform multiple functions ultimately related to the secretion and/or targeting of effector proteins (15, 18) and the yscB-yscM locus encodes type III structural genes (39). VirF itself is under environmental control, and its synthesis is induced when bacteria encounter a 37°C environment (39). Furthermore, recent evidence suggests that temperature regulation of virulence gene expression in Yersinia is modulated through changes in the topology of the virulence plasmid (44). Clearly, AraC homologues are utilized by several pathogenic bacterial species to regulate expression of type III secretion systems and effectors, which are essential virulence determinants for these organisms. An additional AraC homologue, MxiE, has been described in Shigella, although the genes that it may regulate have not yet been identified (2). The purpose of this study was to determine whether MxiE functions as a transcriptional regulator in Shigella flexneri and to identify targets of its regulation. Here we describe an additional level of virulence gene regulation in Shigella which functions only after host cell invasion and identify six virulence plasmid-encoded genes that are activated by MxiE in the intracellular environment.