SummaryMany Gram-negative pathogenic bacteria use a type III secretion (T3S) system to interact with cells of their hosts. Mechanisms controlling the hierarchical addressing of needle subunits, translocators and effectors to the T3S apparatus (T3SA) are still poorly understood. We investigated the function of MxiC, the member of the YopN/InvE/SepL family in the Shigella flexneri T3S system. Inactivation of mxiC led specifically to a deregulated secretion of effectors (including IpaA, IpgD, IcsB, IpgB2, OspD1 and IpaHs), but not of translocators (IpaB and IpaC) and proteins controlling the T3SA structure or activity (Spa32 and IpaD). Expression of effector-encoding genes controlled by the activity of the T3SA and the transcription activator MxiE was increased in the mxiC mutant, as a consequence of the increased secretion of the MxiE antiactivator OspD1. MxiC is a T3SA substrate and its ability to be secreted is required for its function. By using co-purification assays, we found that MxiC can associate with the Spa47 ATPase, which suggests that MxiC might prevent secretion of effectors by blocking the T3SA from the inside. Although with a 10-fold reduced efficiency compared with the wild-type strain, the mxiC mutant was still able to enter epithelial cells.

SummaryThe type III secretion apparatus (T3SA) is a multiprotein complex central to the virulence of many Gram-negative pathogens. Currently, the mechanisms controlling the hierarchical addressing of needle subunits, translocators and effectors to the T3SA are still poorly understood. In Shigella, MxiC is known to sequester effectors within the cytoplasm prior to receiving the activation signal from the needle. However, molecules involved in linking the needle and MxiC are unknown. Here, we demonstrate a molecular interaction between MxiC and the predicted inner-rod component MxiI suggesting that this complex plugs the T3SA entry gate. Our results suggest that MxiIMxiC complex dissociation facilitates the switch in secretion from translocators to effectors. We identified MxiC F206S variant, unable to interact with MxiI, which exhibits a constitutive secretion phenotype although it remains responsive to induction. Moreover, we identified the mxiI Q67A mutant that only secretes translocators, a phenotype that was suppressed by coexpression of the MxiC F206S variant. We demonstrated the interaction between MxiI and MxiC homologues in Yersinia and Salmonella. Lastly, we identified an interaction between MxiC and chaperone IpgC which contributes to understanding how translocators secretion is regulated. In summary, this study suggests the existence of a widely conserved T3S mechanism that regulates effectors secretion.

SummaryThe effectors of enterocyte invasion by Shigella are dependent on a type III secretion system that contains a needle whose length average does not exceed 50 nm. Previously, we reported that Spa32 is required for needle length control as well as to switch substrate specificity from MxiH to Ipa proteins secretion. To identify functional domains of Spa32, 11 truncated variants were constructed and analysed for their capacity (i) to control the needle's length; (ii) to secrete the Ipa proteins; and (iii) to invade HeLa cells. Deletion at either the N-terminus or C-terminus affect Spa32 function in all cases, but Spa32 variants lacking internal residues 37-94 or 130-159 retained full Spa32 function. Similarly, a Spa32 variant obtained by inserting of the YscP's ruler domain retained Spa32 function although it programmed slightly elongated needles. Using the GST pull-down assay, we show that residues 206-246 are required for Spa32 binding to the C-terminus of Spa40, an inner membrane protein required for Ipa proteins secretion. Our data clearly demonstrate that shortening Spa32 affects the length of the needle in a comparable manner to the spa32 mutant, indicating that the control of needle length does not require a molecular ruler mechanism.

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The core Mga (multiple gene activator) regulon of group A Streptococcus (GAS) contains genes encoding proteins involved in adhesion and immune evasion. While all GAS genomes contain genes for Mga and C5a peptidase, the intervening genes encoding M and M-like proteins vary between strains. The genetic make-up of the Mga regulon of GAS was characterized by utilizing a collection of 1,688 GAS genomes that are representative of the global GAS population. Sequence variations were examined with multiple alignments, and the expression of all core Mga regulon genes was examined by quantitative reverse transcription-PCR in a representative strain collection. In 85.2% of the sampled genomes, the Mga locus contained genes encoding Mga, Mrp, M, Enn, and C5a peptidase proteins. These isolates account for 53% of global infections. Only 9.1% of genomes did not contain either an mrp or an enn gene. The pairwise identity within Enn (68.6%) and Mrp (83.2%) protein sequences was higher than within M proteins (44.7%). Gene expression varied between strains tested, but high expression was recorded for all genes in at least one strain. Previous nomenclature issues were clarified with molecular gene definitions. Our findings support a shift in focus in the GAS research field to further consider the role of Mrp and Enn in virulence and vaccine development. IMPORTANCE While the GAS M protein has been the leading vaccine target for decades, the bacteria encode many other virulence factors of interest for vaccine development. In this work, we show that emm-like genes are encoded in a remarkable majority of GAS genomes and expressed at a level similar to that for the emm gene. In collaboration with the U.S. Centers for Disease Control, we developed molecular definitions of the different emm and emm-like gene families. This clarification should abrogate mistyping of strains, especially in the area of whole-genome typing. We have also updated the emm-typing collection by removing emm-like gene sequences and provided in-depth analysis of Mrp and Enn protein sequence structure and diversity.

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The type III secretion apparatus (T3SA) is a central virulence factor of many Gram-negative bacteria. Its overall morphology consists of a cytoplasmic region, inner-and outer-membrane sections and an extracellular needle. In Shigella, the length of the needle is regulated by Spa32. To understand better the role of Spa32 we searched for its interacting partners using a two-hybrid screen in yeast. We found that Spa32 interacts with the 33 C-terminal residues (CC*) of Spa40, a member of the conserved FlhB/YscU family. Using a GST pull-down assay we confirmed this interaction and identified additional interactions between Spa40 and the type III secretion components Spa33, Spa47, MxiK, MxiN and MxiA. Inactivation of spa40 abolished protein secretion and led to needleless structures. Genetic and functional analyses were used to investigate the roles of residues L310 and V320, located within the CC* domain of Spa40, in the assembly of the T3SA. Spa40 cleavage, at the conserved NPTH motif, is required for assembly of the T3SA and for its interaction with Spa32, Spa33 and Spa47. In contrast, unprocessed forms of Spa40 interacted only with MxiA, MxiK and MxiN. Our data suggest that the conformation of the cytoplasmic domain of Spa40 defines the multi-step assembly process of the T3SA. INTRODUCTIONShigella flexneri, the causative agent of bacillary dysentery in humans, is able to enter and to disseminate in epithelial cells by using a specialized Mxi-Spa type III secretion apparatus (T3SA) (Parsot, 2009). The T3SA is widespread among Gram-negative bacteria that are pathogenic for animals and plants or are symbionts (Viprey et al., 1998;Dale et al., 2001Dale et al., , 2002 and is devoted to the injection of virulence effectors into target cells. The T3SA is composed of a cytoplasmic region called 'the bulb', a basal body consisting of a pair of rings that span the inner and outer membranes, and an extracellular needle protruding outside the bacterium. This apparatus is evolutionarily and structurally related to the export machinery of flagellar systems (Macnab, 1999;Blocker et al., 2001). Nine T3SA constituents are related to components of the basal bodies of bacterial flagella (Allaoui et al., 1994;Aizawa, 2001;Blocker et al., 2003;Macnab, 2004), including the innermembrane proteins MxiA (FlhA), Spa9 (FliP), Spa24 (FliR), Spa29 (FliQ) and Spa40 (FlhB), and the cytoplasmic proteins Spa47 (FliI), MxiN (FliH) and Spa33 (FliN) (Kane et al., 2002;Allaoui et al., 1992bAllaoui et al., , 1993bAllaoui et al., , 1995Penno et al., 2006;Jouihri et al., 2003;Andrews & Maurelli, 1992; Sasakawa et al., 1993;Schuch & Maurelli, 2001; MoritaIshihara et al., 2005). Additionally, two cytoplasmic proteins, Spa13 and MxiK, which have no obvious counterpart in the flagellar system, are also implicated in Abbreviations: EPEC, enteropathogenic Escherichia coli; GST, glutathione S-transferase; NC, needle complex; T3S, type III secretion; T3SA, type III secretion apparatus; T3SS, type III secretion system; TEM, transmission electron microscopy.3The...

M and M-like surface proteins from group A Streptococcus (GAS) act as virulence factors and have been used in multiple vaccine candidates. While the M protein has been extensively studied, the two genetically and functionally related M-like proteins, Mrp and Enn, although present in most streptococcal strains have been relatively less characterised. We compile the current state of knowledge for these two proteins, from discovery to recent studies on function and immunogenicity, using the M protein for comparison as a prototype of this family of proteins. We focus on the known interactions between M-like proteins and host ligand proteins, and analyse the genetic data supporting these interactions. We discuss known and possible functions of M-like proteins during GAS infections, and highlight knowledge gaps where further investigation is warranted.