Characterization of a dipartite iron uptake system from uropathogenic Escherichia coli strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His⁴⁴, Met⁹⁰, His⁹⁷, and His¹²⁷, and CuB, a second degenerate octahedral geometry with the addition of Glu⁴⁶. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.     

Biochemical detection of N-Acyl homoserine lactone from biofilm-forming uropathogenic Escherichia coli isolated from urinary tract infection samples

Background:

N-Acyl homoserine lactone (AHL) is found to be the main component of quorum sensing (QS) in Gram-negative bacteria and plays an important role in biofilm formation. Little information is available regarding the role of AHL in biofilm formation in Escherichia coli (E. coli). The purpose of this investigation was to biochemically detect and characterize AHL activity in biofilm-forming uropathogenic E. coli (UPEC) isolated from urine samples of the patients with urinary tract infections (UTIs) in Kerman, Iran.

Methods:

Thirty-five UPEC isolates were obtained from urine samples of the patients with UTIs referred to the Afzalipoor hospital. The isolates were identified by biochemical tests. Biofilm analyses of all the isolates were performed using the microtiter plate method at OD 490nm. N-Acyl homoserine lactone was separated from cell mass supernatants by liquid-liquid extraction (LLE) and analyzed by a colorimetric method. N-Acyl homoserine lactone functional groups were identified by Fourier Transform-Infrared Spectroscopy (FT-IR).

Results:

The biofilm formation assay identified 10 (28.57%) isolates with strong, 16 (45.71%) with moderate, and 9 (25.71%) with weak biofilm activities. The UPEC isolates with strong and weak biofilm activities were subjected to AHL analyses. It was found that isolates with the highest AHL activities also exhibited strong adherence to microplate wells (P≤0.05). Two E. coli isolates with the highest AHL activities were selected for FT-IR spectroscopy. Peaks at 1764.33, 1377.99, and 1242.90 cm^-1 correspond to the C=O bond of the lactone ring, and the N=H and C-O bonds of the acyl chain, respectively.

Conclusion:

We found that many UPEC isolates exhibited strong biofilm formation. The control of this property by AHL may contribute to the pathogenesis of the organism in UTI’s.Key Words: Biofilm, FT-IR, N-acylhomoserine lactone, Uropathogenic Escherichia coli     

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract

Background  

The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model.     

The effects of oxidative stress in urinary tract infection during pregnancy

The purpose of this study was to determine the effect of urinary tract infection (UTI) on antioxidant systems and lipid peroxidation (LPO) levels during pregnancy. We also investigated if these antioxidant systems and LPO levels differed in each trimester. One hundred forty-three nonpregnant women, as a control group, and 77 pregnant women were included in the study. Urine cultures were performed according to standard techniques. Catalase (CAT), superoxide dismutase (SOD), and LPO levels were measured using a spectrophotometer. UTI was observed in 14 of 77 pregnant women and the isolated microorganisms were Escherichia coli, Klebsiella pneumoniae, and Staphylococcus saprophyticus. CAT, SOD, and LPO levels were increased in pregnant women compared with nonpregnant women (P<.01). CAT, SOD activities, and LPO levels were increased from the first trimester to the third trimester in pregnancy without UTI. However, CAT and SOD activities were decreased, LPO levels were increased from the first trimester to the third trimester in pregnancy with UTI (P<.01). Pregnancy causes oxidative stress and also UTI during pregnancy may aggravate oxidative stress.     

Roles of iron acquisition systems in virulence of extraintestinal pathogenic Escherichia coli: salmochelin and aerobactin contribute more to virulence than heme in a chicken infection model

ABSTRACT: BACKGROUND: Avian pathogenic Escherichia coli (APEC) and uropathogenic E. coli (UPEC) are the two main subsets of extraintestinal pathogenic E. coli (ExPEC). Both types have multiple iron acquisition systems, including heme and siderophores. Although iron transport systems involved in the pathogenesis of APEC or UPEC have been documented individually in corresponding animal models, the contribution of these systems during simultaneous APEC and UPEC infection is not well described. To determine the contribution of each individual iron acquisition system to the virulence of APEC and UPEC, isogenic mutants affecting iron uptake in APEC E058 and UPEC U17 were constructed and compared in a chicken challenge model. RESULTS: Salmochelin-defective mutants E058DeltairoD and U17DeltairoD showed significantly decreased pathogenicity compared to the wild-type strains. Aerobactin defective mutants E058DeltaiucD and U17DeltaiucD demonstrated reduced colonization in several internal organs, whereas the heme defective mutants E058DeltachuT and U17DeltachuT colonized internal organs to the same extent as their wild-type strains. The triple mutant DeltachuTDeltairoDDeltaiucD in both E058 and U17 showed decreased pathogenicity compared to each of the single mutants. The histopathological lesions in visceral organs of birds challenged with the wild-type strains were more severe than those from birds challenged with DeltairoD, DeltaiucD or the triple mutants. Conversely, chickens inoculated with the DeltachuT mutants had lesions comparable to those in chickens inoculated with the wild-type strains. However, no significant differences were observed between the mutants and the wild-type strains in resistance to serum, cellular invasion and intracellular survival in HD-11, and growth in iron-rich or iron-restricted medium. CONCLUSIONS: Results indicated that APEC and UPEC utilize similar iron acquisition mechanisms in chickens. Both salmochelin and aerobactin systems appeared to be important in APEC and UPEC virulence, while salmochelin contributed more to the virulence. Heme bounded by ChuT in the periplasm appeared to be redundant in this model, indicating that other periplasmic binding proteins likely contributed to the observed no phenotype for the heme uptake mutant. No differences were observed between the mutants and their wild-type parents in other phenotypic traits, suggesting that other virulence mechanisms compensate for the effect of the mutations.     

Involvement of the Leishmania donovani virulence factor A2 in protection against heat and oxidative stress

Leishmania is an obligate intracellular protozoan parasite that infects cells of the reticulo-endothelial system. Host defences against Leishmania include fever and oxidant production, and the parasite has developed a number of defence mechanisms to neutralize the host response. The Leishmania donovani A2 family of proteins has been shown to be essential for survival in mammalian visceral organs. Here we provide evidence that A2 proteins protect the parasite against host defences, namely heat stress (fever) and oxidative stress. A2 is however unable to protect the cells from endoplasmic reticulum stress induced by dithiothreitol. To downregulate A2 protein expression, L. donovani was transfected with an A2 antisense RNA expressing-vector, resulting in significant reduction of A2 levels. The resulting A2-deficient cells were more sensitive to heat shock and this was associated with increased production of internal oxidants during heat shock. Moreover, axenic amastigotes with downregulated A2 expression had increased internal oxidants and decreased viability following treatment with hydrogen peroxide or a nitric oxide donor when compared to control cells. Overall, these results suggest that A2 protects L. donovani from a variety of stresses, thereby allowing it to survive in the internal organs of the mammalian host and to cause visceral disease.     

Detection of clonal group A Escherichia coli isolates from broiler chickens, broiler chicken meat, community-dwelling humans, and urinary tract infection (UTI) patients and their virulence in a mouse UTI model

Escherichia coli clonal group A isolates cause infections in people. We investigated 158 phylogroup D E. coli isolates from animals, meat, and humans. Twenty-five of these isolates were of clonal group A, and 15 isolates were shown to cause infection in a mouse urinary tract infection (UTI) model. We conclude that clonal group A isolates are found in both broiler chickens and broiler chicken meat and may cause UTI in humans.     

Immunological approach to the pathogenetical role of Escherichia coli adhesive factor "119" in a suckling mouse urinary tract model

In the course of urinary tract infections, suckling mice with maternal anti-pilus ("119") immunity showed a massive protection against a 119+ strain of Escherichia coli. Animals could be protected against urinary tract infection by giving pilus antibody or pilus vaccine shortly after the infection. Results showed the importance of adhesive pili in initiating the urinary tract infection by E. coli.     

Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli

The histone-like protein H-NS is a global regulator in Escherichia coli that has been intensively studied in nonpathogenic strains. However, no comprehensive study on the role of H-NS and its paralogue, StpA, in gene expression in pathogenic E. coli has been carried out so far. Here, we monitored the global effects of H-NS and StpA in a uropathogenic E. coli isolate by using DNA arrays. Expression profiling revealed that more than 500 genes were affected by an hns mutation, whereas no effect of StpA alone was observed. An hns stpA double mutant showed a distinct gene expression pattern that differed in large part from that of the hns single mutant. This suggests a direct interaction between the two paralogues and the existence of distinct regulons of H-NS and an H-NS/StpA heteromeric complex. hns mutation resulted in increased expression of alpha-hemolysin, fimbriae, and iron uptake systems as well as genes involved in stress adaptation. Furthermore, several other putative virulence genes were found to be part of the H-NS regulon. Although the lack of H-NS, either alone or in combination with StpA, has a huge impact on gene expression in pathogenic E. coli strains, its effect on virulence is ambiguous. At a high infection dose, hns mutants trigger more sudden lethality due to their increased acute toxicity in murine urinary tract infection and sepsis models. At a lower infectious dose, however, mutants lacking H-NS are attenuated through their impaired growth rate, which can only partially be compensated for by the higher expression of numerous virulence factors.     

Specificity of immunomodulator secretion in urinary samples in response to infection by alpha-hemolysin and CNF1 bearing uropathogenic Escherichia coli

Escherichia coli are the most common etiological agents of urinary tract infections (UTIs). Uropathogenic E. coli (UPECs) produce specific toxins including the cytotoxic necrotizing factor-1 (CNF1) and the alpha-hemolysin (alpha-Hly). CNF1 triggers, through Rho protein activation, a specific gene response of host cells, which results in the production for instance of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and the macrophage inflammatory protein-3alpha (MIP-3alpha). The alpha hemolysin alpha-Hly also triggers the production of inflammatory mediators. Cnf1 is always associated with alpha-hly in a pathogenicity island conserved among UPECs. Using two complementary approaches we have investigated whether alpha-hly and cnf1 bearing UPECs are associated with a specific type of UTI both in term of pathology and host response. Here we report that UPECs bearing alpha-hly/cnf1 have a prevalence of 50% in UPECs isolated from hemorrhagic UTIs, as compared to 30% in the overall UPEC population. In addition, we observed that MCP-1, and IL-8 to a lower extent, is produced in urine at higher concentrations in UTIs caused by UPECs carrying alpha-hly/cnf1.     

LPS-induced CD53 expression: a protection mechanism against oxidative and radiation stress

The CD53 antigen is a member of the tetraspanin membrane protein family that is expressed in the lymphoid-myeloid lineage. Its biological role remains unknown. Using microarrays, we identified CD53 as one of the principal genes up-regulated by exposure of macrophages to LPS. Northern blot analysis confirmed the induction of CD53 in RAW264.7 macrophages treated with LPS or SNAP (a nitric oxide donor). Cells stably transfected with sense CD53 cDNA had increased levels of intracellular GSH and lower levels of peroxide, and were more resistant to H2O2 and to UVB irradiation. Cells harboring antisense CD53 had the opposite properties. We propose that the induction of CD53 is a major mechanism by which macrophages protect themselves against LPS-induced oxidative stress and UVB irradiation.     

The role of the natural polyamine putrescine in defense against oxidative stress in Escherichia coli

Putrescine up-regulated, in a concentration-dependent manner, the expression levels of the oxyR and katG genes of Escherichia coli cells exposed to hydrogen peroxide. Its stimulatory effect was more pronounced under conditions of strong oxidative stress. 1,4-Diamino-2-butanone, a specific inhibitor of putrescine synthesis, also inhibited oxyR expression under oxidative stress. When added to inhibited cells, putrescine relieved this inhibitory effect. Addition of putrescine to E. coli cultures exposed to oxidative stress led to increased cell survival.