Latefa Biskri

Erythromycin Esterase Gene ere(A) Is Located in a Functional Gene Cassette in an Unusual Class 2 Integron

ABSTRACT The gene ere(A) of the plasmid pIP1100 is larger than originally reported and is organized as an integron gene cassette. The ere(A) gene cassette carries its own promoter and is propagated by a class 2 integron with an insertion sequence element, IS1, inserted upstream of the intI2 gene. The mobility of the ere(A) cassette has been demonstrated.

MxiC is secreted by and controls the substrate specificity of the Shigella flexneri type III secretion apparatus

Many 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 anti‐activator 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.

Comparative Study of Class 1 Integron and Vibrio cholerae Superintegron Integrase Activities

Superintegrons (SIs) and multiresistant integrons (MRIs) have two main structural differences: (i) the SI platform is sedentary, while the MRI platform is commonly associated with mobile DNA elements and (ii) the recombination sites (attC) of SI gene cassette clusters are highly homogeneous, while those of MRI cassette arrays are highly variable in length and sequence. In order to determine if the latter difference was correlated with a dissimilarity in the recombination activities, we conducted a comparative study of the integron integrases of the class 1 MRI (IntI1) and the Vibrio cholerae SI (VchIntIA). We developed two assays that allowed us to independently measure the frequencies of cassette deletion and integration at the cognate attI sites. We demonstrated that the range of attC sites efficiently recombined by VchIntIA is narrower than the range of attC sites efficiently recombined by IntI1. Introduction of mutations into the V. cholerae repeats (VCRs), the attC sites of the V. cholerae SI cassettes, allowed us to map positions that affected the VchIntIA and IntI1 activities to different extents. Using a cointegration assay, we established that in E. coli, attI1-x-VCR recombination catalyzed by IntI1 was 2,600-fold more efficient than attIVch-x-VCR recombination catalyzed by VchIntIA. We performed the same experiments in V. cholerae and established that the attIVch-x-VCR recombination catalyzed by VchIntIA was 2,000-fold greater than the recombination measured in E. coli. Taken together, our results indicate that in the V. cholerae SI, the substrate recognition and recombination reactions mediated by VchIntIA might differ from the class 1 MRI paradigm.

IpgB1 and IpgB2, two homologous effectors secreted via the Mxi-Spa type III secretion apparatus, cooperate to mediate polarized cell invasion and inflammatory potential of Shigella flexenri

Type III secretion systems (T3SS) are present in many pathogenic gram-negative bacteria and mediate the translocation of bacterial effector proteins into host cells. Here, we report the phenotypic characterization of S. flexneri ipgB1 and ipgB2 mutants, in which the genes encoding the IpgB1 and IpgB2 effectors have been inactivated, either independently or simultaneously. Like IpgB1, we found that IpgB2 is secreted by the T3SS and its secretion requires the Spa15 chaperone. Upon infection of semi-confluent HeLa cells, the ipgB2 mutant exhibited the same invasive capacity as the wild-type strain and the ipgB1 mutant was 50% less invasive. Upon infection of polarised Caco2-cells, the ipgB2 mutant did not show a significant defect in invasion and the ipgB1 mutant was slightly more invasive than the wild-type strain. Entry of the ipgB1 ipgB2 mutant in polarized cells was reduced by 70% compared to the wild-type strain. Upon infection of the cornea in Guinea pigs, the ipgB2 mutant exhibited a wild-type phenotype, the ipgB1 mutant was hypervirulent and elicited a more pronounced proinflammatory response, while the ipgB1 ipgB2 mutant was highly attenuated. The attenuated phenotype of the ipgB1 ipgB2 mutant was confirmed using a murine pulmonary model of infection and histopathology and immunochemistry studies.

Interplay between predicted inner-rod and gatekeeper in controlling substrate specificity of the type III secretion system.

The type III secretion apparatus (T3SA) is a multi‐protein 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 MxiI–MxiC complex dissociation facilitates the switch in secretion from translocators to effectors. We identified MxiCF206S variant, unable to interact with MxiI, which exhibits a constitutive secretion phenotype although it remains responsive to induction. Moreover, we identified the mxiIQ67A mutant that only secretes translocators, a phenotype that was suppressed by coexpression of the MxiCF206S 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.

Transcriptional slippage controls production of type III secretion apparatus components in Shigella flexneri.

During transcription, series of approximately 9 As or Ts can direct RNA polymerase to incorporate into the mRNA nucleotides not encoded by the DNA, changing the reading frame downstream from the slippage site. We detected series of 9 or 10 As in spa13, spa33 and mxiA encoding type III secretion apparatus components. Analysis of cDNAs indicated that transcriptional slippage occurs in spa13, mxiA and spa33. Changes in the reading frame were confirmed by using plasmids carrying slippage sites in the 5′ part of lacZ. Slippage is required for production of Spa13 from two overlapping reading frames and should lead to production of truncated MxiA and Spa33 proteins. Complementation of spa13 and mxiA mutants with plasmids carrying altered sites indicated that slippage in spa13 is required for assembly of the secretion apparatus and that slippage sites in spa13 and mxiA have not been selected to encode Lys residues or to produce two proteins endowed with different activities. The presence of slippage sites decreases production of Spa13 by 70%, of MxiA and Spa33 by 15% and of Spa32 (encoded downstream from spa13) by 50%. These results suggest that transcriptional slippage controls protein production by reducing the proportion of mRNA translated into functional proteins.

The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri

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.

Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact.

Type III secretion apparatus (T3SA) are complex nanomachines that insert a translocation pore into the host cell membrane through which effector proteins are injected into the cytosol. In Shigella, the pore is inserted by a needle tip complex that also controls secretion. IpaD is the key protein that rules the composition of the tip complex before and upon cell contact or Congo red (CR) induction. However, how IpaD is involved in secretion control and translocon insertion remains not fully understood. Here, we report the phenotypic analysis of 20 10‐amino acids deletion variants all along the coiled‐coil and the central domains of IpaD (residues 131–332). Our results highlight three classes of T3S phenotype; (i) wild‐type secretion, (ii) constitutive secretion of all classes of effectors, and (iii) constitutive secretion of translocators and early effectors, but not of late effectors. Our data also suggest that the composition of the tip complex defines both the T3SA inducibility state and late effectors secretion. Finally, we shed light on a new aspect regarding the contact of the needle tip with cell membrane by uncoupling the Shigella abilities to escape macrophage vacuole, and to insert the translocation pore or to invade non‐phagocytic cells.

Characterization of SfPgdA, a Shigella flexneri peptidoglycan deacetylase required for bacterial persistence within polymorphonuclear neutrophils

Peptidoglycan deacetylases protect the Gram-positive bacteria cell wall from host lysozymes by deacetylating peptidoglycan. Sequence analysis of the genome of Shigella flexneri predicts a putative polysaccharide deacetylase encoded by the plasmidic gene orf185, renamed here SfpgdA. We demonstrated a peptidoglycan deacetylase (PGD) activity with the purified SfPgdA in vitro. To investigate the role SfPgdA in virulence, we constructed a SfpgdA mutant and studied its phenotype in vitro. The mutant showed an increased sensitivity to lysozyme compared to the parental strain. Moreover, the mutant was rapidly killed by polymorphonuclear neutrophils (PMNs). Specific substitution of histidines residues 120 and 125, located within the PGD catalytic domain, by phenylalanine abolished SfPgdA function. SfPgdA expression is controlled by PhoP. Mutation of phoP increases sensitivity to lysozyme compared to the SfpgdA mutant. Here, we confirmed that SfPgdA expression is enhanced under low magnesium concentration and not produced by the phoP mutant. Ectopic expression of SfPgdA in the phoP mutant restored lysozyme resistance and parental bacterial persistence within PMNs. Together our results indicate that PG deacetylation mechanism likely contributes to Shigella persistence by subverting detection by the host immune system.

Shigella Stands up to the Challenge of Adhesion

The invasion process of S.xa0flexneri is well characterized, but mechanisms underlying this bacteriums adhesion to host cells have remained obscure. In this issue of Cell Host & Microbe, Brotcke Zumsteg etxa0al. (2014) report a surprising role for the Shigella virulence factor IcsA (VirG) as an adhesin.