hlyD Genes Research Articles

Molecular typing and virulence characteristics of Escherichia coli strains isolated from hospital and community acquired urinary tract infections.

The main causes of hospital- and community-acquired urinary tract infections (UTIs) are a group of Escherichia coli (E. coli) strains with multiple virulence factors known as uropathogenic E. coli. One hundred E. coli isolates from the urine specimens of hospital- and community-acquired UTI patients were characterized based on their virulence factors and genetic relatedness using PCR and RAPD‒PCR, respectively. Among all, the traT (71%), sitA (64%), ompT (54%), malX (49%), ibeA (44%), tsh (39%), hlyD (18%) and cnf1 (12%) genes had the highest to lowest frequencies, respectively. There was no significant difference between the frequency of tested virulence genes in E. coli isolates from inpatients and outpatients. The frequency of the hlyD gene was significantly greater in E. coli isolates from patients hospitalized in gynecology, dermatology and intensive care unit (ICU) wards than in those from other wards. Eight virulence gene patterns were common among the isolates of inpatients in different wards of the same hospital, of which five patterns belonged to the isolates of inpatients in the same ward. More E. coli isolates with similar virulence gene patterns and greater genetic similarity were found in female patients than in male patients. The analysis of the RAPD‒PCR dendrograms revealed more genetic similarities among the E. coli isolates from inpatients than among those from outpatients. Our findings indicate the presence of a wide variety of virulence factors in E. coli isolates and the possibility of spreading the same clones in different wards of the hospital.

Frequency of hlyA, hlyB, hlyC and hlyD genes in uropathogenic Escherichia coli isolated from UTI patients in Shiraz.

Background and objectives: One of the most important causes of urinary tract infections (UTI) is Escherichia coli. The infection is mainly due to the uropathogenic strain (UPEC), which has key virulence factors, including hemolysis. In this study, we evaluated the frequency of hlyA, hlyB, hlyC and hlyD genes in UPEC strains isolated from clinical samples from Shiraz city, Iran.Materials and methods: 130 urine samples with suspected UTI were collected from Shiraz medical centers and cultured on blood agar and EMB media. Colonies were then characterized by biochemical methods. The genomes were extracted and the presence of hemolysis genes was detected by polymerase chain reaction (PCR) using hly gene specific primers and 16S rRNA. Drug resistance was assessed by using 10 antibiotic disks in the disk diffusion method, according to CLSI criteria.Results: Out of the 130 collected UTI samples, 100 were identified as UPECs. Within isolates, the hlyD gene had the highest frequency – 95% – and hlyC had the lowest, with 23%. The frequencies of hlyA and hlyB genes were calculated as 50% and 43%, respectively. The rates of antibiotic resistance to Azithromycin, Ampicillin, Cefotaxime, Nalidixic Acid, Tetracycline, Trimethoprim-Sulfamethoxazole, Cefepime, Aztreonam, Gentamicin, and Nitrofurantoin were 95%, 86%, 68%, 66%, 65%, 64%, 51%, 46%, 44%, 14%, respectively. 98% of these isolates belonged to the MDR group.Conclusion: This study shows diversity of hemolysis virulence factor in UPECs and unique UPEC drug resistance that would indicate a high antibiotic use in the general population.

Contribution of Lysinibacillus sphaericus hemolysin and chitin-binding protein in entomopathogenic activity against insecticide resistant Aedes aegypti

Given its toxicity against culicid larvae, Lysinibacillus sphaericus is used for the biological control of mosquitoes such as Culex sp. and Anopheles sp. The toxicity factors currently reported for L. sphaericus include the Binary toxin, Mtx toxins, and the S-layer. However, Aedes aegypti is refractory to the Binary toxin, the most toxic larvicidal protein of L. sphaericus. Until now, there are no evidences of the hemolytic and chitinolytic capacity of L. sphaericus. Herein, the expression of the hemolysin D (hlyD) and the chitin-binding protein genes of L. sphaericus III(3)7, OT4b.25, and 2362 was quantified. Gene expression was assessed 24 and 48h after field-collected and Rockefeller A. aegypti larvae were fed with the bacteria. The hlyD gene showed the highest expression at 24h whilst the expression of the chitin-binding protein gene increases at 48h. The highest hlyD expression was seen in the III(3)7 strain and the highest chitin-binding protein gene expression was in the 2362 strain. The consortium of L. sphaericus III(3)7 and 2362 showed the highest expression of both genes being with field-collected and Rockefeller larvae. The results suggest that hemolysin D and the chitin-binding protein can be two novel toxic elements involved in the entomopathogenic activity of L. sphaericus. These proteins, along with the other L. sphaericus toxins, make this bacterium a suitable alternative to replace the chemical insecticides used for the control of A. aegypti populations.

Prevalence of virulence factors and phylogenetic characterization of uropathogenic Escherichia coli causing urinary tract infection in patients with and without diabetes mellitus.

We aimed to determine the virulence factor profile and phylogenetic grouping of uropathogenic Escherichia coli (UPEC) causing urinary tract infection (UTI) in patients with and without diabetes mellitus (DM). A total of 280 UPEC were collected from the urine samples of patients with DM (n=126) and patients suffering from non-DM UTIs (n=154). All isolates were subjected to phenotypic and genotypic virulence factor profile and phylogenetic grouping. There was no significant difference in phenotypic virulence factors of UPEC causing UTI from patients with and without DM; alpha-haemolysin (DM=52, 41.2%; non-DM=69, 44.8%), mannose resistant haemagglutination (DM=6, 36.2%; non-DM=64, 41.5%), biofilm production (DM=33.3%; non-DM=25.9%), serum resistance (DM=27, 21.4%; non-DM=46, 29.8%), cell surface hydrophobicity (DM=22, 17.1%; non-DM=18, 11.6%) and mannose sensitive hemagglutnation (DM=18, 14.2%; non-DM=29, 18.8%). Among the genotypic virulence markers, papC gene was most prevalent in both patients with DM (n=65, 51.6%) and without DM (n=88, 57.1%), followed by hlyD gene (DM=36, 28.6%; non-DM=52, 33.8%). Only cnf-1 gene was observed to be significantly associated (p<0.05) with the non-DM status (n=35, 22.7%) than DM (n=15, 11.9%). Irrespective of the diabetic status, majority of the UPEC isolates (approximately 50%) belonged to phylogenetic group B2 predominantly harboring the virulence genes papC, hlyD and cnf-1. This study demonstrates that there may not be a differential selection of virulence properties of UPEC causing UTI in patients with DM and in the general population.

Phylogenetic grouping and pathotypic comparison of urine and fecal Escherichia coli isolates from children with urinary tract infection

The aim of this study was to investigate the phylogenetic background and to assess hlyD (involved in the secretion of haemolysin A) and intI1 (encoding a class 1 integrase) in Escherichia coli isolates derived from urinary and fecal specimens. A total of 200 E. coli isolates was collected from patients presenting with urinary tract infection (UTI) during September 2009 to September 2010 and screened for hlyD and intI1 genes by polymerase chain reaction (PCR). Phylogenetic analysis showed that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D) and that uropathogenic E. coli (UPEC) isolates mainly belong to groups B2 (54%) and D (34%) whereas group A (44%) and D (26%) are predominant among commensal E. coli isolates. In this study, hlyD was present in 26% of UPEC and 2% of commensal E. coli isolates. However, hemolytic activity was detected for 42% of UPEC and 6% of commensal E. coli isolates (p < 0.05). intI1 gene was more frequently expressed in UPEC (24%) in comparison with commensal E. coli isolates (12%). Resistance to aztreonam, co-trimoxazole and cefpodoxime were frequently found among UPEC isolates whereas commensal E. coli isolates were commonly resistant to co-trimoxazole, nalidixic acid and cefotaxime. Concluding, a considerable difference between UPEC and commensal E. coli isolates was observed regarding their phylogenetic groups, presence of class 1 integron and hlyD gene, hemolysin activity and resistance pattern. The detection of class 1 integrons and hlyD gene was higher among UPEC compared with commensal E. coli isolates. These findings may contribute for a better understanding of the factors involved in the pathogenesis of UPEC.

VerotoxigenicEscherichia colifrom animals, humans and foods: who's who?

Verocytotoxigenic (shigatoxigenic) and enterohaemorrhagic Escherichia co/i, VTEC (STEC) and EHEC, produce a toxin active on Vero cells in vitro. VTEC and EHEC have been isolated from humans and different animal species, mainly ruminants and pigs. The verocytotoxins, also named shiga toxins (Stx), are active in vivo on the endothelial cells of the blood vessels of the gastro-intestinal mucosa, the kidneys, the brain, and other tissues of humans and piglets, leading to fluid leakage or haemorrhages. Conversely, their role in diseases of young ruminants remains unclear. Adult ruminants can also act as asymptomatic carriers of VTEC and EHEC strains similar to those causing diseases in humans. And they are incriminated as an important source of direct or indirect contamination of humans by the most famous EHEC strain belonging to the O157:H7 serotype, through faecal contamination of either foods of animal origin, or other foodstuffs (fruit, vegetables, etc.), or the environment. But dozens of non-O157 human and ruminant VTEC and EHEC strains with similar general and virulence-associated properties, have been described, whose epidemiology is much less well understood. The purpose of this review manuscript is to describe and compare the properties of human, ruminant and food VTEC and EHEC strains.

Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain.

Clostridium difficile causes pseudomembranous colitis through the action of Rho-modifying proteins, toxins A and B. Antibodies directed against C. difficile toxin A prevent or limit C. difficile-induced colitis. We engineered plasmid pETR14, containing the hlyB and hlyD genes of the Escherichia coli hemolysin operon, to express a fusion protein containing 720 amino acid residues from the nontoxic, receptor-binding, carboxy terminus of C. difficile toxin A and the secretion signal of E. coli hemolysin A. We introduced pETR14 into Vibrio cholerae and found that the toxin A-HlyA fusion protein was secreted by a number of V. cholerae strains and recognized by both monoclonal and polyclonal anti-C. difficile toxin A antibodies. We introduced pETR14 into an attenuated V. cholerae strain, O395-NT, and inoculated rabbits orally with this construct. Colonization studies disclosed that the V. cholerae vector containing pETR14 was recoverable from rabbit ilea up to 5 days after oral inoculation. Vaccination produced significant systemic anti-C. difficile toxin A immunoglobulin G and anti-V. cholerae vibriocidal antibody responses. Vaccination also produced significant protection against toxin A in an ileal loop challenge assay, as assessed by determination of both fluid secretion and histological changes. These results suggest that the hemolysin system of E. coli can be used successfully in V. cholerae vector strains to effect secretion of large heterologous antigens and that a V. cholerae vector strain secreting a nontoxic, immunogenic portion of C. difficile toxin A fused to the secretion signal of E. coli HlyA induces protective systemic and mucosal immunity against this toxin.

Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain EDL 933

In this study, we determined the nucleotide sequence of the 5.4-kb SalI restriction fragment of the recombinant plasmid pEO40-1, cloned from the large plasmid of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain EDL 933. This revealed two open reading frames which shared approximately 60% homology to the hlyC and hlyA genes of the E. coli alpha-hemolysin (alpha-hly) operon. We termed these genes EHEC-hlyA and EHEC-hlyC to distinguish them from the alpha-hly genes. Preliminary sequence analysis indicated that another open reading frame homolog to the hlyB gene is located close to the 3' end of EHEC-hlyA. The predicted molecular masses of the EHEC-hlyA and EHEC-hlyC gene products were 107 and 19.9 kDa, respectively. The EHEC hemolysin protein (EHEC-Hly) was not secreted into the culture supernatant by the strain EDL 933. However, hemolytic activity was found in the broth culture supernatant after transforming EDL 933 with the recombinant plasmid pRSC6 carrying the hlyB and hlyD genes from the E. coli alpha-hemolysin operon. The EHEC hemolysin was precipitated and used as an antigen for immunoblot analysis. This demonstrated that 19 of 20 reconvalescent-phase serum samples from patients with hemolytic uremic syndrome reacted specifically with the antigen; conversely, only 1 of 20 control serum samples demonstrated reactivity. To investigate the prevalence of EHEC hemolysin genes in diarrheagenic E. coli, a PCR was developed to specifically detect EHEC-hlyA. All Shiga-like toxin-producing O157 strains and 12 of 25 Shiga-like toxin-producing non-O157 strains were PCR positive; strains of other categories of diarrheagenic E. coli were PCR negative. All PCR-positive strains hybridized with the CVD 419 probe. We found the CVD 419 probe to be identical to the 3.4-kb HindIII fragment of plasmid pEO40 carrying most of the EHEC-hlyA gene and a part of the putative EHEC-hlyB gene. In this study, the newly discovered EHEC hemolysin was shown to be responsible for the enterohemolytic phenotype and demonstrated to be related but not identical to alpha-hemolysin. The EHEC hemolysin appears to have clinical importance because it occurs in all O157 strains tested and is reactive to sera of patients with hemolytic uremic syndrome.

Processing by OmpT of fusion proteins carrying the HlyA transport signal during secretion by the Escherichia coli hemolysin transport system.

A fusion gene (ces-hlyAs) was constructed by ligating the genetic information for the C-terminal 60 amino acids (hlyAs) of Escherichia coli hemolysin (HlyA) to the ces gene for a cholesterol esterase/lipase (CE) from a Pseudomonas species. Part (about 30%) of the expressed fusion protein CE-HlyAs was secreted in E. coli carrying hlyB and hlyD genes. Following the insertion between the reporter gene and hlyAs of a linker sequence that contains the information for potential cleavage sites for the outer membrane protease OmpT, two different fusion proteins (PhoA-HlyAs and CE-HlyAs) were shown to be cleaved by OmpT between the two parts during HlyB/HlyD-mediated secretion. Processed PhoA and CE accumulated in the supernatant. The efficiency of cleavage by OmpT was considerably improved by increased ompT gene dose. It was further shown that OmpT preferentially recognizes potential cleavage sites within the linker sequence.

Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of the Pasteurella leukotoxin by Escherichia coli.

Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli. The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli. Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD.

Secretion of thePasteurellaleukotoxin byEscherichia coli

Nucleic acid sequence analysis has indicated that the leukotoxin determinant from Pasteurella haemolytica is related to the hemolysin determinant from E. coli. The cloning and expression in E. coli of the lktCA genes has been previously reported, but the existence of leukotoxin secretory genes equivalent to hlyBD has not been documented. In this report we demonstrate that a 4.0 kb segment of P. haemolytica genomic DNA distal to the lktA gene, when expressed in trans to the previous cloned lktCA genes, allow the synthesis and secretion of active leukotoxin from E. coli. Complementation analysis using the cloned hlyB and hlyD genes indicates that this secretory locus derived from P. haemolytica contains two genes which we designate, by analogy, lktB and lktD.