Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth.

Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S. flexneri that were cultured with, but did not invade, Henle cells (extracellular). Compared to in vitro and extracellular bacteria, intracellular bacteria had increased levels of proteins required for invasion and cell-to-cell spread, including Ipa, Mxi, and Ics proteins. Changes in metabolic pathways in response to the intracellular environment also were evident. There was an increase in glycogen biosynthesis enzymes, altered expression of sugar transporters, and a reduced amount of the carbon storage regulator CsrA. Mixed acid fermentation enzymes were highly expressed intracellularly, while tricarboxylic acid (TCA) cycle oxidoreductive enzymes and most electron transport chain proteins, except CydAB, were markedly decreased. This suggested that fermentation and the CydAB system primarily sustain energy generation intracellularly. Elevated levels of PntAB, which is responsible for NADPH regeneration, suggested a shortage of reducing factors for ATP synthesis. These metabolic changes likely reflect changes in available carbon sources, oxygen levels, and iron availability. Intracellular bacteria showed strong evidence of iron starvation. Iron acquisition systems (Iut, Sit, FhuA, and Feo) and the iron starvation, stress-associated Fe-S cluster assembly (Suf) protein were markedly increased in abundance. Mutational analysis confirmed that the mixed-acid fermentation pathway was required for wild-type intracellular growth and spread of S. flexneri. Thus, iron stress and changes in carbon metabolism may be key factors in the S. flexneri transition from the extra- to the intracellular milieu.

RyhB, an iron-responsive small RNA molecule, regulates Shigella dysenteriae virulence.

Regulation of bacterial gene expression by small RNA (sRNA) molecules is an increasingly recognized phenomenon but one that is not yet fully understood. We show that the sRNA RyhB suppresses several virulence-associated phenotypes of Shigella dysenteriae, a causative agent of bacillary dysentery in humans. The virulence genes repressed by S. dysenteriae RyhB include those encoding the type III secretion apparatus, its secreted effectors, and specific chaperones. Suppression of Shigella virulence occurs via RyhB-dependent repression of the transcriptional activator VirB, leading to reduced expression of genes within the VirB regulon. Efficient repression of virB is mediated by a single-stranded region of RyhB that is distinct from the region required for repression of Shigella sodB. Regulation of virB by RyhB implicates iron as an environmental factor contributing to the complex regulation of Shigella virulence determinants.

Reduced renal Na+-K+-Cl- co-transporter activity and inhibited NKCC2 mRNA expression by Leptospira shermani: from bed-side to bench.

BACKGROUND: Renal involvement is common in leptospirosis. Interstitial nephritis with interstitial oedema and mononuclear cellular infiltration are the usual findings. Clinically, non-oligouric acute renal failure, hypokalaemia and sodium wasting appear frequently in leptospirosis. The outer membrane protein from leptospira has been thought to be responsible for the disorder. However, the exact mechanisms of renal involvement are still unclear. METHODS: To address these questions, we first performed detailed in vivo clearance tests in three patients with leptospirosis (Leptospira shermani) and hypokalaemia to define the tubular lesion. These tests indicated a defective Na(+)-K(+)-Cl(-) co-transporter and a poor response to furosemide infusion. We performed further in vitro studies in a model of medullary thick ascending limb (mTAL) cultured cells derived from normal mouse. RESULTS: Outer membrane protein extract from L.shermani (0.3 microg/ml) inhibited Na(+)-K(+)-Cl(-) co-transporter activity in mTAL cells (control, 10.15+/-0.52; L.shermani, 6.47+/-0.47 nmol/min/mg protein). The basolateral Na(+)-K(+) ATPase remained intact. Reverse transcription-polymerase chain reaction (RT-PCR) further revealed that the outer membrane protein extract from L.shermani downregulated Na(+)-K(+)-Cl(-) co-transporter (mNKCC2) mRNA expression. These changes were dose dependent and could be reversed by the antibody against outer membrane protein extract from L.shermani. Experiments with a less pathogenic strain of leptospira (L.bratislava) and Escherichia coli did not affect Na(+)-K(+)-Cl(-) co-transporter activity. CONCLUSIONS: We conclude that L.shermani leptospirosis downregulates mNKCC2 mRNA expression and inhibits Na(+)-K(+)-Cl(-) co-transporter activity in TAL cells. These alterations may explain the observed electrolyte disorders in leptospirosis.

Contribution of the Shigella flexneri Sit, Iuc, and Feo iron acquisition systems to iron acquisition in vitro and in cultured cells.

Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.

Identification of two Shigella flexneri chromosomal loci involved in intercellular spreading.

The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.

Intercellular spread of Shigella flexneri through a monolayer mediated by membranous protrusions and associated with reorganization of the cytoskeletal protein vinculin.

The spread of Shigella flexneri in a monolayer of infected Henle and HeLa cells was studied by using immunofluorescence and electron microscopy. Infected cells produced numerous bacterium-containing membranous protrusions up to 18 microns in length that penetrated adjacent cells and were subsequently phagocytosed. Fluorescence staining of actin and vinculin in infected cells with phalloidin and monoclonal antibody to vinculin, respectively, demonstrated that the protrusions containing the bacteria consisted of these cytoskeletal proteins. Actin accumulated predominantly at the poles of bacteria distal to the tip of protrusions and appeared as trails extending back towards the host cell cytoplasm. Vinculin, however, was distributed uniformly around the bacteria and throughout the protrusion. A profound rearrangement of vinculin occurred in Henle and HeLa cells following infection with shigellae: whereas in uninfected cells it was distributed mainly around the cell periphery, in infected cells it concentrated mainly around clusters of bacteria in the cytoplasm. This suggests a possible involvement of the vinculin cytoskeletal protein in the intercellular spread of shigellae during an infection.

The distribution of leptospires in the kidney tubules of some British wild mammals.

Histological examination of 69 pairs of infected kidneys from 12 species of Rodentia, Lagomorpha, Carnivora and Insectivora revealed that leptospires were confined mainly to the proximal and distal convoluted tubule, were less often found in the thick loop of Henle and only rarely in the collecting duct. On no occasion were the organisms present in the thin loop of Henle. Preliminary observations on the relationship of leptospires to tubule epithelium indicate some degree of physical attachment. It is suggested that the avoidance of the thin loop of Henle might be a reflection of its structural properties.