Laurence du Merle

Monoclonal antibodies for identification of Borrelia afzelii sp. nov. associated with late cutaneous manifestations of Lyme borreliosis.

Borrelia isolates associated with Lyme borreliosis were previously divided into 3 genospecies, B. burgdorferi sensu stricto, B. garinii and group VS461, on the basis of DNA homology. B. burgdorferi sensu stricto and B. garinii were identified by monoclonal antibodies (MAbs), H3TS and D6 respectively, but no MAbs were available to identify group VS461. Two MAbs were produced, I 17.3 and J 8.3 which reacted with OspB and OspA proteins, respectively, of strains belonging to group VS461, which should be named B. afzelii sp. nov. 24 strains were assigned to B. afzelii sp. nov., 11 of them being isolated from skin lesions, 6 from acrodermatitis chronica atrophicans (ACA) and 5 from erythema chronicum migrans (ECM). Although quite unknown in the USA, ACA has frequently been reported in northern Europe where B. afzelii sp. nov. is commonly isolated. This study documents the involvement of B. afzelii sp. nov. as a specific aetiological agent of ACA.

Binding of Escherichia coli adhesin AfaE to CD55 triggers cell-surface expression of the MHC class I-related molecule MICA

MICA are distant homologs of MHC class I molecules expressed in the normal intestinal epithelium. They are ligands of the NKG2D activating receptor expressed on most γδ T cells, CD8+ αβ T cells, and natural killer cells and therefore play a critical role in innate immune responses. We investigated MICA cell-surface expression on infection of epithelial cell lines by enteric bacteria and show here that MICA expression can be markedly increased by bacteria of the diffusely adherent Escherichia coli diarrheagenic group. This effect is mediated by the specific interaction between bacterial adhesin AfaE and its cellular receptor, CD55, or decay-accelerating factor. It is extremely rapid after AfaE binding, consistent with a stress-induced signal. MICA induction on epithelial cells triggered IFN-γ release by the NKG2D expressing natural killer cell line NKL. This host–bacteria interaction pathway could play a role in the pathogenesis of inflammatory bowel disease, a condition that implicates a bacterial trigger in genetically susceptible individuals. This was supported by the increased MICA expression at the surface of epithelial cells in colonic biopsies from Crohns disease-affected patients compared with controls.

Renal Collecting Duct Epithelial Cells React to Pyelonephritis-Associated Escherichia coli by Activating Distinct TLR4-Dependent and -Independent Inflammatory Pathways

TLR4 plays a central role in resistance to pyelonephritis caused by uropathogenic Escherichia coli (UPEC). It has been suggested that renal tubule epithelial cells expressing TLRs may play a key role in inflammatory disorders and in initiating host defenses. In this study we used an experimental mouse model of ascending urinary tract infection to show that UPEC isolates preferentially adhered to the apical surface of medullary collecting duct (MCD) intercalated cells. UPEC-infected C3H/HeJ (Lpsd) mice carrying an inactivating mutation of tlr4 failed to clear renal bacteria and exhibited a dramatic slump in proinflammatory mediators as compared with infected wild-type C3H/HeOuJ (Lpsn) mice. However, the level of expression of the leukocyte chemoattractants MIP-2 and TNF-α still remained greater in UPEC-infected than in naive C3H/HeJ (Lpsd) mice. Using primary cultures of microdissected Lpsn MCDs that expressed TLR4 and its accessory molecules MD2, MyD88, and CD14, we also show that UPECs stimulated both a TLR4-mediated, MyD88-dependent, TIR domain-containing adaptor-inducing IFN-β-independent pathway and a TLR4-independent pathway, leading to bipolarized secretion of MIP-2. Stimulation by UPECs of the TLR4-mediated pathway in Lpsn MCDs leads to the activation of NF-κB, and MAPK p38, ERK1/2, and JNK. In addition, UPECs stimulated TLR4-independent signaling by activating a TNF receptor-associated factor 2-apoptosis signal-regulatory kinase 1-JNK pathway. These findings demonstrate that epithelial collecting duct cells are actively involved in the initiation of an immune response via several distinct signaling pathways and suggest that intercalated cells play an active role in the recognition of UPECs colonizing the kidneys.

Recognition of the cellular beta1-chain integrin by the bacterial AfaD invasin is implicated in the internalization of afa-expressing pathogenic Escherichia coli strains.

The afa operons from Escherichia coli associated with extra‐intestinal and intestinal infections have been characterized and the AfaD protein has been shown to be involved in the low internalization of laboratory strains expressing the afa‐3 operon. The aim of this study was to determine the role of the AfaD invasin during the interaction of pathogenic E. coli with epithelial cells. We show that AfaD is implicated in the entry of a clinical isolate into both HeLa and undifferentiated Caco‐2 cells. Once in the cytoplasm of these cells, the bacteria formed inclusions in which they were able to survive for at least 72 h. Internalization assays using polystyrene beads coated with His6‐tagged purified AfaD (rAfaD) demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for afa‐positive strains. Consistent with the previous observation that an antibody blockade involving anti‐α5β1 integrin abolishes bacterial internalization, we show here that the entry of rAfaD‐coated beads was dependent on the production and accessibility of β1 integrins on the cells. The AfaD proteins belong to a family of invasins that are at least 45% identical. Despite their differences, the recombinant rAfaD‐III and rAfaD‐VIII proteins both bound to β1 integrins. Our results suggest that β1 integrin is a common receptor for AfaD invasins and that additional AfaD‐type‐specific receptors exist.

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells.

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder. The AL511 clinical isolate adhered to mouse collecting duct mpkCCDcl4 cells, used as a model of renal cell invasion, and was able to enter and persist within these cells. Previous studies have shown that bacterial flagella play an important role in host urinary tract colonization, but the role of flagella in the interaction of E. coli with renal epithelial cells remains unclear. An analysis of the ability of E. coli AL511 mutants to invade renal cells showed that flagellin played a key role in bacterial entry. Both flagellum filament assembly and the motor proteins MotA and MotB appeared to be required for E. coli AL511 uptake into collecting duct cells. These findings indicate that pyelonephritis‐associated E. coli strains may invade renal collecting duct cells and that flagellin may act as an invasin in this process.

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem.

The solution structure of the invasive tip complex from Afa/Dr fibrils.

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three‐dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.

Repression of invasion genes and decreased invasion in a high-level fluoroquinolone-resistant Salmonella typhimurium mutant.

Background Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability. Methodology/Principal Findings Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 µg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant. Conclusions/Significance In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.

An in silico model for identification of small RNAs in whole bacterial genomes: characterization of antisense RNAs in pathogenic Escherichia coli and Streptococcus agalactiae strains

Characterization of small non-coding ribonucleic acids (sRNA) among the large volume of data generated by high-throughput RNA-seq or tiling microarray analyses remains a challenge. Thus, there is still a need for accurate in silico prediction methods to identify sRNAs within a given bacterial species. After years of effort, dedicated software were developed based on comparative genomic analyses or mathematical/statistical models. Although these genomic analyses enabled sRNAs in intergenic regions to be efficiently identified, they all failed to predict antisense sRNA genes (asRNA), i.e. RNA genes located on the DNA strand complementary to that which encodes the protein. The statistical models enabled any genomic region to be analyzed theorically but not efficiently. We present a new model for in silico identification of sRNA and asRNA candidates within an entire bacterial genome. This model was successfully used to analyze the Gram-negative Escherichia coli and Gram-positive Streptococcus agalactiae. In both bacteria, numerous asRNAs are transcribed from the complementary strand of genes located in pathogenicity islands, strongly suggesting that these asRNAs are regulators of the virulence expression. In particular, we characterized an asRNA that acted as an enhancer-like regulator of the type 1 fimbriae production involved in the virulence of extra-intestinal pathogenic E. coli.

The Flagella of an Atypical Enteropathogenic Escherichia coli Strain Are Required for Efficient Interaction with and Stimulation of Interleukin-8 Production by Enterocytes In Vitro

ABSTRACT The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells. The role of flagella in aEPEC 1711-4 adhesion, invasion, and IL-8 production was investigated by performing the same experiments with an isogenic aEPEC mutant unable to produce flagellin (FliC), the flagellum protein subunit. We demonstrated that this mutant (fliC mutant) had a marked decrease in the ability to adhere to T84 cells and invade both T84 and Caco-2 cells in gentamicin protection assays and by transmission electron microscopy. In addition, the aEPEC 1711-4 fliC mutant had a reduced ability to stimulate IL-8 production by Caco-2 cells in early (3-h) but not in late (24-h) infections. Our findings demonstrate that flagella of aEPEC 1711-4 are required for efficient adhesion, invasion, and early but not late IL-8 production in intestinal epithelial cells in vitro.