A system for identifying post-invasion functions of invasion genes: requirements for the Mxi-Spa type III secretion pathway of Shigella flexneri in intercellular dissemination.

Invasion and intercellular spread are hallmarks of Shigella pathogenicity. Invasion of the eukaryotic cell cytosol requires a type III secretion system (Mxi-Spa) and its cognate set of secreted Ipa invasins. Once intracellular, the IcsA protein directs a form of actin-based motility that helps to drive intracellular bacterial movement, formation of cellular protrusions and cell-to-cell spread. Work in our laboratory has focused on identifying additional factors required for this intercellular form of dissemination. In this study, we sought to identify novel contributions of the type III secretion pathway to post-invasion-specific processes, distinct from its previously characterized roles in invasion. Studies of post-invasion Ipa and Mxi-Spa functions are complicated by an absolute requirement for these virulence proteins in invasion. To circumvent this problem, we developed a system called TIER (for test of intracellular expression requirements), whereby specific ipa, mxi or spa loci are transiently expressed before infection of tissue culture cell monolayers (thus supporting invasion), but then repressed after invasion in the intracellular environment. Such invasive type III secretion mutants (called TIER mutants) were severely restricted in their ability to spread intercellularly and form plaques in confluent tissue culture cell monolayers. Intercellular spread defects were associated with the repression of most type III pathway components examined, including structural (MxiM and Spa33), secreted effector (IpaB, IpaC and IpaD) and regulatory elements (VirF and VirB). A kinetic analysis of bacterial growth in L2 cell monolayers showed that each of the TIER mutants was defective with respect to long-term intracellular proliferation and viability. Examination of TIER mutant-infected monolayers by electron microscopy revealed that the type III pathway was required for a late step in intercellular spread - bacterial escape from protrusion-derived, double-membrane-bound vacuoles. The TIER mutants were eventually degraded in a process involving vacuolar acidification. Based on these findings, we propose that Ipa secretion via Mxi-Spa is required in the protrusion vacuole for double-membrane lysis.

Establishment of unipolar localization of IcsA in Shigella flexneri 2a is not dependent on virulence plasmid determinants.

Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type S. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigella and E. coli.

Identification of a rough strain of Escherichia coli O157:H7 that produces no detectable O157 antigen.

MA6, an O157:H7-like strain, did not react with most anti-O157 kits examined; however, it had the rfbE gene that is essential for O157 expression and carried O157:H7 virulence factors. Lipopolysaccharide analysis showed that MA6 is a rough strain that does not produce the O157 antigen, but genetically, it belongs in the O157:H7 clonal group.

Effect of O side-chain length and composition on the virulence of Shigella flexneri 2a.

IcsA of Shigella flexneri is required for intercellular spread and is located in the outer membrane at one pole of the bacterium, where it catalyses the polymerization of host-cell actin. The formation of the a tin tail provides the force to move the bacterium in a unidirectional manner through the host-cell cytoplasm. We have previously demonstrated that rough lipopolysaccharide (LPS) mutants of S. flexneri 2a are avirulent and cannot form plaques in tissue-culture monolayers. This inability to form plaques is associated with non-polar localization of IcsA and loss of host-cell membrane-protrusion formation ("fireworks'). To define the minimal LPS structure required for fireworks formation, we constructed a strain of S. flexneri (BS497) that contains a mutation in rfc, encoding the O side-chain polymerase, and a strain, BS520, that possesses a defective O side-chain ligase due to a mutation in rfaL. BS497 produces a LPS that consists of a core with one repeat unit of the O side-chain, while BS520 produces a LPS consisting of a complete core with no O side-chain. BS497 remained invasive but did not form fireworks or plaques in tissue-culture monolayers and was negative in the Serény test. BS520 was invasive, generated reduced numbers of short fireworks, and made tiny plaques, but it was negative in the Serény test. Analysis of BS497 with anti-IcsA antibody demonstrated that IcsA was distributed over the entire cell surface. The distribution of IcsA on the surface of BS520 was predominantly unipolar, with some trail-back of IcsA label along the sides of the bacterium. A similar pattern was seen when infected monolayers were stained for polymerized actin. These results suggest that both the presence and the length of the O side-chain are important in the proper localization or maintenance of IcsA at the pole which subsequently affects the ability to form actin tails and produce fireworks. This reduced ability to form actin tails and fireworks results in a decreased ability of Shigella to move into adjacent host cells. To determine if the sugar composition of the O side-chain is important in the ability to form fireworks, the rfb region of S. flexneri 2a was replaced with the rfb region from Escherichia coli serotype O8 or O25. Both hybrids were invasive, formed plaques, and gave positive Serény reactions. These results suggest that, unlike LPS length, the sugar composition of the O side-chain is not a critical requirement for the proper localization of IcsA and efficient intercellular movement.

Avirulence of rough mutants of Shigella flexneri: requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane.

Mutations in the lipopolysaccharide (LPS) of Shigella spp. result in attenuation of the bacteria in both in vitro and in vivo models of virulence, although the precise block in pathogenesis is not known. We isolated defined mutations in two genes, galU and rfe, which directly affect synthesis of the LPS of S. flexneri 2a, in order to determine more precisely the step in virulence at which LPS mutants are blocked. The galU and rfe mutants invaded HeLa cells but failed to generate the membrane protrusions (fireworks) characteristic of intracellular motility displayed by wild-type shigellae. Furthermore, the galU mutant was unable to form plaques on a confluent monolayer of eucaryotic cells and the rfe mutant generated only tiny plaques. These observations indicated that the mutants were blocked in their ability to spread from cell to cell. Western immunoblot analysis of expression of IcsA, the protein essential for intracellular motility and intercellular spread, demonstrated that both mutants synthesized IcsA, although they secreted less of the protein to the extracellular medium than did the wild-type parent. More strikingly, the LPS mutants showed aberrant surface localization of IcsA. Unlike the unipolar localization of IcsA seen in the wild-type parent, the galU mutant expressed the protein in a circumferential fashion. The rfe mutant had an intermediate phenotype in that it displayed some localization of IcsA at one pole while also showing diffuse localization around the bacterium. Given the known structures of the LPS of wild-type S. flexneri 2a, the rfe mutant, and the galU mutant, we hypothesize that the core and O-antigen components of LPS are critical elements in the correct unipolar localization of IcsA. These observations indicate a more precise role for LPS in Shigella pathogenesis.

Genetic basis of pyocin resistance in Neisseria gonorrhoeae.

The genetic basis for pyocin resistance in Neisseria gonorrhoeae 1291d, 1291e, and FA5100 was determined by Southern blot and DNA sequence analyses. The genes defective in these strains are present as single copies in the gonococcal chromosome. The mutant regions of 1291d, 1291e, and FA5100 were amplified by the PCR. Sequence analysis of the mutant regions demonstrated that strain 1291d contains a 12-bp deletion that results in the loss of four amino acids in phosphoglucomutase, while strain 1291e contains a point mutation that results in the change of an uncharged glycine residue to a charged glutamic acid residue in the same protein. FA5100 contains a nonsense mutation in the gene encoding heptosyltransferase II. The gene previously described as lsi-1 was shown to complement an rfaF mutation in Salmonella typhimurium and has been renamed rfaF.

Role of phosphoglucomutase in lipooligosaccharide biosynthesis in Neisseria gonorrhoeae.

A region of pSG30 that complements the pyocin-derived gonococcal lipooligosaccharide (LOS) mutants 1291d and 1291e was characterized by DNA sequence analysis and an open reading frame of 1,380 bases was identified that is 89% similar and 56% identical over 452 amino acids to the algC gene product from Pseudomonas aeruginosa that encodes phosphomannomutase. Enzymatic analysis of gonococcal crude protein extracts demonstrated that pSG30 encodes phosphoglucomutase (PGM) and phosphomannomutase activity. This activity is absent in 1291d and 1291e but is restored upon introduction of pSG30. PGM encoded by pSG34, a subclone of pSG30, was able to complement Escherichia coli PGM1, a strain deficient in PGM, as determined by bacteriophage C21 plaque formation. A revertant of 1291d that binds monoclonal antibody 2-1-L8 (specific for a 3.6-kDa LOS component) was isolated. The construction of a site-specific deletion of this region in the chromosome of 1291 confirms the role of this open reading frame in LOS biosynthesis.

Cloning of a gonococcal DNA sequence that complements the lipooligosaccharide defects of Neisseria gonorrhoeae 1291d and 1291e.

An isogenic set of gonococcal lipooligosaccharide (LOS) mutants derived from pyocin treatment of Neisseria gonorrhoeae 1291 was used to identify cloned gonococcal DNA fragments. A gene bank from N. gonorrhoeae 1291c chromosomal DNA was made in pLEE10, a shuttle vector that replicates in the gonococcus and Escherichia coli. A plasmid (pSG30) that could transform the LOS mutants 1291d and 1291e to reactivity with monoclonal antibody 3F11 and to production of an LOS component with migration identical to that of the parent, 1291, was identified. pSG30 contains a 9-kb EcoRI fragment. Curing studies indicate that pSG30 encodes gene products that affect LOS biosynthesis in trans. Subcloning identified a 2.6-kb HincII fragment (pSG38) that retained the ability to modify the LOS of 1291d and 1291e. The DNA regions involved in modification of 1291d and 1291e were named lsi-4 and lsi-5, respectively. The region of pSG38 that was involved in LOS modification was further localized by the construction of exonuclease III deletion plasmids. Transformation of these constructs identified a 750-bp fragment that retains the ability to modify 1291e and a 540-bp fragment which retains the ability to modify 1291d.