Role of attP in integrase-mediated integration of the Shigella resistance locus pathogenicity island of Shigella flexneri.
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ABSTRACT:
SUBMITTER: Turner SA
PROVIDER: S-EPMC353151 | biostudies-other | 2004 Mar
REPOSITORIES: biostudies-other
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Project description:In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.
| S-EPMC95444 | biostudies-literature
Project description:Iron uptake systems which are critical for bacterial survival and which may play important roles in bacterial virulence are often carried on mobile elements, such as plasmids and pathogenicity islands (PAIs). In the present study, we identified and characterized a ferric dicitrate uptake system (Fec) in Shigella flexneri serotype 2a that is encoded by a novel PAI termed the Shigella resistance locus (SRL) PAI. The fec genes are transcribed in S. flexneri, and complementation of a fec deletion in Escherichia coli demonstrated that they are functional. However, insertional inactivation of fecI, leading to a loss in fec gene expression, did not impair the growth of the parent strain of S. flexneri in iron-limited culture media, suggesting that S. flexneri carries additional iron uptake systems capable of compensating for the loss of Fec-mediated iron uptake. DNA sequence analysis showed that the fec genes are linked to a cluster of multiple antibiotic resistance determinants, designated the SRL, on the chromosome of S. flexneri 2a. Both the SRL and fec loci are carried on the 66,257-bp SRL PAI, which has integrated into the serX tRNA gene and which carries at least 22 prophage-related open reading frames, including one for a P4-like integrase. This is the first example of a PAI that carries genes encoding antibiotic resistance and the first report of a ferric dicitrate uptake system in Shigella.
| S-EPMC98729 | biostudies-literature
Project description:In recent years, multidrug resistance of Shigella strains associated with genetic elements like pathogenicity islands, have become a public health problem. The Shigella resistance locus pathogenicity island (SRL PAI) of S. flexneri 2a harbors a 16Kbp region that contributes to the multidrug resistance phenotype. However, there is not much information about other functions such as metabolic, physiologic or ecological ones. For that, wild type S. flexneri YSH6000 strain, and its spontaneous SRL PAI mutant, 1363, were used to study the contribution of the island in different growth conditions. Interestingly, when both strains were compared by the Phenotype Microarrays, the ability to metabolize D-aspartic acid as a carbon source was detected in the wild type strain but not in the mutant. When D-aspartate was added to minimal medium with other carbon sources such as mannose or mannitol, the SRL PAI-positive strain was able to metabolize it, while the SRL PAI-negative strain did not. In order to identify the genetic elements responsible for this phenotype, a bioinformatic analysis was performed and two genes belonging to SRL PAI were found: orf8, coding for a putative aspartate racemase, and orf9, coding for a transporter. Thus, it was possible to measure, by an indirect analysis of racemization activity in minimal medium supplemented only with D-aspartate, that YSH6000 strain was able to transform the D-form into L-, while the mutant was impaired to do it. When the orf8-orf9 region from SRL island was transformed into S. flexneri and S. sonnei SRL PAI-negative strains, the phenotype was restored. Although, when single genes were cloned into plasmids, no complementation was observed. Our results strongly suggest that the aspartate racemase and the transporter encoded in the SRL pathogenicity island are important for bacterial survival in environments rich in D-aspartate.
| S-EPMC6980539 | biostudies-literature
Project description:Virulence plasmid (VP) acquisition was a key step in the evolution of Shigella from a non-pathogenic Escherichia coli ancestor to a pathogenic genus. In addition, the co-evolution and co-ordination of chromosomes and VPs was also a very important step in the evolutionary process. To investigate the cross-talk between VPs and bacterial chromosomes, we analyzed the expression profiles of protein complexes and protein monomers in three wild-type Shigella flexneri strains and their corresponding VP deletion mutants. A non-pathogenic wild-type E. coli strain and mutant E. coli strains harboring three Shigella VPs were also analyzed. Comparisons showed that the expression of chromosome-encoded proteins GadA/B and AtpA/D, which are associated with intracellular proton flow and pH tuning of bacterial cells, was significantly altered following acquisition or deletion of the VP. The acid tolerance of the above strains was also compared, and the results confirmed that the presence of the VP reduced the bacterial survival rate in extremely acidic environments, such as that in the host stomach. These results further our understanding of the evolution from non-pathogenic E. coli to Shigella, and highlight the importance of co-ordination between heterologous genes and the host chromosome in the evolution of bacterial species.
| S-EPMC5404508 | biostudies-literature
Project description:Non-human primates (NHPs) for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants isolation and antibiotic testing of enteric pathogens before treating macaques with quinolones prophylactically or therapeutically.
| S-EPMC5844971 | biostudies-literature
Project description:The she gene of Shigella flexneri 2a, which also harbors the internal enterotoxin genes set1A and set1B (F. R. Noriega, GenBank accession no. U35656, 1995) encodes a homolog of the virulence-related immunoglobulin A (IgA) protease-like family of secreted proteins, Tsh, EspC, SepA, and Hap, from an avian pathogenic Escherichia coli, an enteropathogenic E. coli, S. flexneri 5, and Haemophilus influenzae, respectively. To investigate the possibility that this locus was carried on a larger deletable element, the S. flexneri 2a YSH6000T she gene was insertionally disrupted by allelic exchange using a Tn10-derived tetAR(B) cassette. Then, to detect loss of the she locus, the tetracycline-resistant derivative was plated onto fusaric acid medium to select for tetracycline-sensitive revertants, which were observed to arise at a frequency of 10(-5) to 10(-6). PCR and pulsed-field gel electrophoresis analysis confirmed loss of the she::tetAR(B) locus in six independent tetracycline-sensitive isolates. Sample sequencing over a 25-kb region flanking she identified four insertion sequence-like elements, the group II intron-like sequence Sf.IntA, and the 3' end of a second IgA protease-like homolog, sigA, lying 3.6 kb downstream and in an orientation inverted with respect to she. The deletion was mapped to chromosomal NotI fragment A and determined to have a size of 51 kb. Hybridization with flanking probes confirmed that at least 17.7 kb of the 51-kb deletable element was unique to the seven she+ strains investigated, supporting the conclusion that she lay within a large pathogenicity island. The method described in this study, termed island probing, provides a useful tool to further the study of pathogenicity islands in general. Importantly, this approach could also be of value in constructing safer live attenuated bacterial vaccines.
| S-EPMC175661 | biostudies-literature
Project description:ObjectiveTo investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China.MethodsA total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004-2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP).ResultsResistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (CipR) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6-32 mg/L, 8-32 mg/L, and 8-24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser83Leu, Asp87Asn/Gly) and single mutation in parC gene (Ser80Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp87Asn) and China (Asp87Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness.ConclusionsEmergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.
| S-EPMC4100904 | biostudies-literature
Project description:Shigella flexneri has become a significant public health concern accounting for the majority of shigellosis cases worldwide. Even though a multitude of efforts is being made into the development of a vaccine to prevent infections, the absence of a licensed global vaccine compels us to enormously depend on antibiotics as the major treatment option. The extensive-unregulated use of antibiotics for treatment along with natural selection in bacteria has led to the rising of multidrug-resistance Shigella strains. Out of the various mechanisms employed by bacteria to gain resistance, efflux transporters are considered to be one of the principal contributors to antimicrobial resistance. The small multidrug-resistance family consists of unique small proteins that act as efflux pumps and are involved in extruding various antimicrobial compounds. The present study aims to demonstrate the role of an efflux transporter YnfA belonging to the SMR family and its functional involvement in promoting antimicrobial resistance in S. flexneri. Employing various genetic, computational, and biochemical techniques, we show how disrupting the YnfA transporter, renders the mutant Shigella strain more susceptible to some antimicrobial compounds tested in this study, and significantly affects the overall transport activity of the bacteria against ethidium bromide and acriflavine when compared with the wild-type Shigella strain. We also assessed how mutating some of the conserved amino acid residues of YnfA alters the resistance profile and efflux activity of the mutant YnfA transporter. This study provides a functional understanding of an uncharacterized SMR transporter YnfA of Shigella.
| S-EPMC9295541 | biostudies-literature
Project description:Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.
| S-EPMC7979695 | biostudies-literature
Project description:In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.
| S-EPMC97446 | biostudies-literature