Hafida Fsihi

Characterization of Reemerging Chikungunya Virus

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host.

VirR, a response regulator critical for Listeria monocytogenes virulence

Signature‐tagged mutagenesis (STM) was used to identify new genes involved in the virulence of the Gram‐positive intracellular pathogen Listeria monocytogenes. One of the mutants isolated by this technique had the transposon inserted in virR, a gene encoding a putative response regulator of a two‐component system. Deletion of virR severely decreased virulence in mice as well as invasion in cell‐culture experiments. Using a transcriptomic approach, we identified 12 genes regulated by VirR, including the dlt‐operon, previously reported to be important for L. monocytogenes virulence. However, a strain lacking dltA, was not as impaired in virulence as the ΔvirR strain, suggesting a role in virulence for other members of the vir regulon. Another VirR‐regulated gene is homologous to mprF, which encodes a protein that modifies membrane phosphatidyl glycerol with l‐lysine and that is involved in resistance to human defensins in Staphylococcus aureus. VirR thus appears to control virulence by a global regulation of surface components modifications. These modifications may affect interactions with host cells, including components of the innate immune system. Surprisingly, although controlling the same set of genes as VirR, the putative cognate histidine kinase of VirR, VirS, encoded by a gene located three genes downstream of virR, was shown not to be essential for virulence. By monitoring the activity of VirR with a GFP reporter construct, we showed that VirR can be activated independently of VirS, for example through a mechanism involving variations in the level of intracellular acetyl phosphate. In silico analysis of the VirR‐regulated promoters revealed a VirR DNA‐binding consensus site and specific interaction between purified VirR protein and this consensus sequence was demonstrated by gel mobility shift assays. This study identifies a second key virulence regulon in L. monocytogenes, after the prfA regulon.

FbpA, a novel multifunctional Listeria monocytogenes virulence factor

Listeria monocytogenes is a Gram‐positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature‐tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E‐cadherin. fbpA encodes a 570‐amino‐acid polypeptide that has strong homologies to atypical fibronectin‐binding proteins. FbpA binds to immobilized human fibronectin in a dose‐dependent and saturable manner and increases adherence of wild‐type L. monocytogenes to HEp‐2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co‐immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin‐binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA‐rpmH‐dnaA‐dnaN‐recF‐orf‐gyrB‐gyrA, is the same as in other Gram‐positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA‐dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

The Mycobacterium leprae genome: systematic sequence analysis identifies key catabolic enzymes, ATP‐dependent transport systems and a novel polA locus associated with genomic variability

In the framework of the mycobacterial genome sequencing project, a continuous 37049bp sequence from the Mycobacterium leprae chromosome has been determined. Computer analysis revealed 10 complete open reading frames, and nine of their products show similarity to known proteins. Seven of these were identified as the enzyme isocitrate lyase, two P‐type ATPase cation transporters, two AMP‐binding proteins, the ribosomal protein S1, and DNA polymerase I. Interestingly, the polA gene, encoding DNA polymerase, is flanked by two inverted copies of a new class of the M. leprae specific repetitive sequence, RLEP, and this structure resembles a transposable element. A second copy of this element was found at another locus in the genome, but the two copies were not present in equal amounts and could not be found in all isolates of M. leprae. This is the first evidence for genomic variability in the leprosy bacillus and might ultimately be useful for developing a molecular test capable of distinguishing between strains of M. leprae.

Gene arrangement and organization in a ∼ 76 kb fragment encompassing the oriC region of the chromosome of Mycobacterium leprae

A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.

Direct inhibition of NF-κB activation by peptide targeting the NOA ubiquitin binding domain of NEMO

Aberrant and constitutive NF-κB activation are frequently reported in numerous tumor types, making its inhibition an attractive target for the treatment of certain cancers. NEMO (NF-κB essential modulator) is the crucial component of the canonical NF-κB pathway that mediates IκB kinase (IKK) complex activation. IKK activation resides in the ability of the C-terminal domain of NEMO to properly dimerize and interact with linear and K63-linked polyubiquitin chains. Here, we have identified a new NEMO peptide inhibitor, termed UBI (ubiquitin binding inhibitor) that derives from the NOA/NUB/UBAN ubiquitin binding site located in the CC2-LZ domain of NEMO. UBI specifically inhibits the NF-κB pathway at the IKK level in different cell types stimulated by a variety of NF-κB signals. Circular dichroïsm and fluorescence studies showed that UBI exhibits an increased α-helix character and direct, good-affinity binding to the NOA-LZ region of NEMO. We also showed that UBI targets NEMO in cells but its mode of inhibition is completely different from the previously reported LZ peptide (herein denoted NOA-LZ). UBI does not promote dissociation of NEMO subunits in cells but impairs the interaction between the NOA UBD of NEMO and polyubiquitin chains. Importantly, we showed that UBI efficiently competes with the in vitro binding of K63-linked chains, but not with linear chains. The identification of this new NEMO inhibitor emphasizes the important contribution of K63-linked chains for IKK activation in NF-κB signaling and would provide a new tool for studying the complex role of NF-κB in inflammation and cancer.

CHAPTER 16 – Listeria monocytogenes

This chapter focuses mainly on L. monocytogenes invasion of nonphagocytic cells and describe the bacterial determinants as well as their eukaryotic receptors (when identified) involved in the infectious process. L. monocytogenes is widespread in nature and can be commonly found in soil, decaying vegetation, sewage, and wastewater. Because of the multiple ecological niches in which L . monocytogenes can be isolated, the origins of the human infection have long been uncertain, but epidemiologic studies provided evidence for a food borne origin for both outbreaks and sporadic episodes of listeriosis. During the third trimester of gestation, cell-mediated immunity is thought to be impaired predisposing pregnant women to develop listerial bacteremia. Symptoms are not severe and resemble influenza-like illness often associated with myalgias, arthralgias, headache, and backache. For unknown reasons, CNS infection is extremely rare during pregnancy. In conclusion, many properties of Listeria monocytogenes have attracted creative research programs. It is hoped that, in the end, they may help to completely eradicate a disease that is still responsible for many sporadic cases of food borne infection or epidemics with dramatic consequences for patients.