waaC Genes Research Articles

Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

The Inactivation of LPS Biosynthesis Genes in E. coli Cells Leads to Oxidative Stress.

Impaired lipopolysaccharide biosynthesis in Gram-negative bacteria results in the “deep rough” phenotype, which is characterized by increased sensitivity of cells to various hydrophobic compounds, including antibiotics novobiocin, actinomycin D, erythromycin, etc. The present study showed that E. coli mutants carrying deletions of the ADP-heptose biosynthesis genes became hypersensitive to a wide range of antibacterial drugs: DNA gyrase inhibitors, protein biosynthesis inhibitors (aminoglycosides, tetracycline), RNA polymerase inhibitors (rifampicin), and β-lactams (carbenicillin). In addition, it was found that inactivation of the gmhA, hldE, rfaD, and waaC genes led to dramatic changes in the redox status of cells: a decrease in the pool of reducing NADPH and ATP equivalents, the concentration of intracellular cysteine, a change in thiol homeostasis, and a deficiency in the formation of hydrogen sulfide. In “deep rough” mutants, intensive formation of reactive oxygen species was observed, which, along with a lack of reducing agents, such as reactive sulfur species or NADPH, leads to oxidative stress and an increase in the number of dead cells in the population. Within the framework of modern ideas about the role of oxidative stress as a universal mechanism of the bactericidal action of antibiotics, inhibition of the enzymes of ADP-heptose biosynthesis is a promising direction for increasing the effectiveness of existing antibiotics and solving the problem of multidrug resistance.

PLEIOTROPIC EFFECT OF MUTATION IN THE GENES INVOLVED
 IN THE BIOSYNTHESIS OF LIPOPOLYSACCHARIDES OF ERWINIA
 AMYLOVORA PHYTOPATHOGENIC BACTERIA

In this study, we constructed Erwinia amylovora deletion mutant by waaC, waaD, waaF, waaL, wabK and wabM genes, coding enzymes involved in the synthesis of lipopolysaccharides of the bacteria outer membrane, and investigated the phenotypic manifestation of the resulting mutation. It was demonstrated that the resulting mutant has an increased ability to autoaggregation and biofilms formation, but is characterized by the reduced mobility and levan exopolysaccharide production. In addition, the deletion of genes of lipopolysaccharides biosynthesis led to the loss of the mutant strain virulence. The data obtained indicate the most important role the lipopolysaccharide layer plays in the virulence of E. amylovora bacteria.

Stress Tolerance and Virulence-Related Roles of Lipopolysaccharide in Burkholderia glumae.

The lipopolysaccharide (LPS) composed of lipid A, core, and O-antigen is the fundamental constituent of the outer membrane in gram-negative bacteria. This study was conducted to investigate the roles of LPS in Burkholderia glumae, the phytopathogen causing bacterial panicle blight and seedling rot in rice. To study the roles of the core oligosaccharide (OS) and the O-antigen region, mutant strains targeting the waaC and the wbiFGHI genes were generated. The LPS profile was greatly affected by disruption of the waaC gene and slight reductions were observed in the O-antigen region following wbiFGHI deletions. The results indicated that disruption in the core OS biosynthesis-related gene, waaC, was associated with increased sensitivity to environmental stress conditions including acidic, osmotic, saline, and detergent stress, and to polymyxin B. Moreover, significant impairment in the swimming and swarming motility and attenuation of bacterial virulence to rice were also observed in the waaC-defective mutant. The motility and virulence of O-antigen mutants defective in any gene of the wbiFGHI operon, were not significantly different from the wild-type except in slight decrease in swimming and swarming motility with wbiH deletion. Altogether, the results of present study indicated that the LPS, particularly the core OS region, is required for tolerance to environmental stress and full virulence in B. glumae. To our knowledge, this is the first functional study of LPS in a plant pathogenic Burkholderia sp. and presents a step forward toward full understanding of B. glumae pathogenesis.

Genotypic Analysis of the Virulence and Antibiotic Resistance Genes in Campylobacter species in silico.

Campylobacter species is responsible for 400-500 million diarrhea cases worldwide every year. Emergence of antibiotic resistance has further complicated the scenario. A wide range of virulence factors and resistance genes are present in Campylobacter species and it is hypothesized there are genotypic variations in the prevalence of these genes. The study was conducted to investigate the presence of virulence and antibiotic resistance genes as well as to investigate difference in prevalence rate based on genotype through in silico tools. Among 26 species studied, sixteen isolates (61.54%) had the cdtB gene that breaks the double helix bonds. The cdtA genes were detected in ten (38.46%) C. jejuni strains while fifty percent (n=13) isolates harbored the cdtC genes. Ten isolates that harboured all three adjacent cdt genes were most toxigenic. The lipo-oligosaccharides associated genes, cgtB and wlaN, responsible for β-1,3 galactosyltransferase production, were found in 7.69% and 30.77% of the isolates, respectively. About 57.69% isolates expressed waaC genes. Invasion protein ciaB, outer membrane phospholipase A pldA and IV secretory protein virB11 were found in 53.85%, 34.62% and 7.69% of the isolates, respectively. Six isolates (23.08%) expressed both tetO and tetA genes while one isolate expressed only tetA resistance gene. Seven isolates (26.92%) had changes in gyrB genes that conferred the fluoroquinolone resistance. In silico PFGE typing found that genotype 3 contained all the virulence genes except cgtB gene while genotype 3 and 4 contained mutated gyrB gene. Genotype 1 and 5 contained no virulence and antibiotic resistance genes. Our data helps to predict the possibility of the presence of virulence and antibiotic resistance genes and helps to select appropriate antibiotic that are more efficacious.

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis of Three Lipooligosaccharide-Associated Genes of Campylobacter jejuni and Campylobacter coli Isolated From Animal Samples

Background: Several genetic mechanisms are used by Campylobacter spp. to achieve pathogenesis. One of the involved virulence factors is lipooligosacharide-associated genes, which are related to ganglioside mimicry by Campylobacter species. Objectives: The current study was conducted to determine the genetic diversity of 3 LOS-associated genes among Campylobacter jejuni and C. coli isolated from animal fecal samples. Methods: One hundred broiler, cattle, and sheep fecal C. jejuni and C. coli isolates, which had been collected previously from Shiraz slaughterhouses and had been formerly identified by polymerase chain reaction (PCR) reactions, were used in the present study. Campylobacter species were subjected to detect wlaN, cgtB, and waaC genes. The PCR products of three LOS-genes were subjected for restriction fragment length polymorphism (RFLP) using HindIII and AluI restriction enzymes in separate reactions. The most prevalent RFLP patterns in the combination of 2 enzymes were subjected for sequencing and sequence analysis software. Results and Conclusions: Patterns of RFLP for PCR products of all 69 wlaN and 29 cgtB genes were similar yet among 86 waaC gene PCR products, 6 different RFLP patterns were obtained. In conclusion, PCR-RFLP analysis demonstrated considerable variation in gene content and overall sequence heterogeneity in the animal-associated C. jejuni and C. coli LOS biosynthesis genes.

Putting on the brakes: Bacterial impediment of wound healing.

The epithelium provides a crucial barrier to infection, and its integrity requires efficient wound healing. Bacterial cells and secretomes from a subset of tested species of bacteria inhibited human and porcine corneal epithelial cell migration in vitro and ex vivo. Secretomes from 95% of Serratia marcescens, 71% of Pseudomonas aeruginosa, 29% of Staphylococcus aureus strains, and other bacterial species inhibited epithelial cell migration. Migration of human foreskin fibroblasts was also inhibited by S. marcescens secretomes indicating that the effect is not cornea specific. Transposon mutagenesis implicated lipopolysaccharide (LPS) core biosynthetic genes as being required to inhibit corneal epithelial cell migration. LPS depletion of S. marcescens secretomes with polymyxin B agarose rendered secretomes unable to inhibit epithelial cell migration. Purified LPS from S. marcescens, but not from Escherichia coli or S. marcescens strains with mutations in the waaG and waaC genes, inhibited epithelial cell migration in vitro and wound healing ex vivo. Together these data suggest that S. marcescens LPS is sufficient for inhibition of epithelial wound healing. This study presents a novel host-pathogen interaction with implications for infections where bacteria impact wound healing and provides evidence that secreted LPS is a key factor in the inhibitory mechanism.

Occurrence of virulence genes and strain diversity of thermophilic campylobacters isolated from cattle and sheep faecal samples

The objective of this study was to investigate the genotypic characteristics of Campylobacter isolates in Shiraz, Iran. A total of 40 Campylobacter isolates including 20 C. jejuni and 20 C. coli were recovered from both cattle and sheep faeces by cultivation methods. The isolates were identified on the basis of polymerase chain reaction (PCR) detection of 16SrRNA and multiplex PCR to determine two species. For confirmed isolates, PCR was carried out for the presence of virulence genes using specific primers. Other than verifying the genetic diversity of thermophilic Campylobacter isolates, flaA PCR-RFLP was performed. Results showed the high prevalence (100%) of the cadF gene and three genes associated with cytolethal distending toxin (CDT). Plasmid virB11 gene was not found in any Campylobacter isolate, and dissimilarities and discrepancies occurred in pldA, iamA, wlaN, waaC and cgtB genes. Among the 40 Campylobacter isolates studied, nine different types were defined by flaA-typing. Results indicated genetic diversity among Campylobacter isolates recovered from cattle and sheep faecal samples. Findings showed the potential ability of C. jejuni and C. coli with cattle and sheep origins to cause infection in humans.

A novel strategy to isolate cell-envelope mutants resistant to phage infection: bacteriophage mEp213 requires lipopolysaccharides in addition to FhuA to enter Escherichia coli K-12

We have developed a direct and efficient strategy, based on a three-step method, to select bacterial cell-envelope mutants resistant to bacteriophage infection. Escherichia coli K-12 strain W3110 underwent classical transposon mutagenesis followed by replica plating and selection for mutants resistant to infection by coliphage mEp213. To verify that phage resistance was due to mutations in the cell envelope, we transformed host cells with the viral genome using electroporation and selected those in which virions were subsequently detected in the supernatant. Among the nine mutants resistant to coliphage infection that we selected, six were in the fhuA gene, two were mutated in the waaC gene, and one was mutated in the gmhD gene. The latter two gene products are involved in the synthesis of lipopolysaccharide (LPS). The efficiency of plating and adsorption of phage mEp213 was affected in these mutants. We verified that LPS is required for the efficient infection of phage λ as well. We propose that this mutation-and-selection strategy can be used to find host factors involved in the initial steps of phage infection for any cognate pair of phage and bacteria.

Kdo-(2 → 8)-Kdo-(2 → 4)-Kdo but not Kdo-(2 → 4)-Kdo-(2 → 4)-Kdo is an acceptor for transfer of L-glycero-α-D-manno-heptose by Escherichia coli heptosyltransferase I (WaaC)

Early steps in the biosynthesis of lipopolysaccharide (LPS) involve the transfer of 3-deoxy-alpha-D-manno-oct-2-ulopyranosonic acid (Kdo) to lipid A. Whereas Kdo transferases (WaaA) of Escherichia coli generate a (2 --> 4)-linked Kdo disaccharide, Chlamydiae contain tri- or tetra-functional WaaA generating oligosaccharides with (2 --> 8)- and (2 --> 4)-linkages between Kdo. It has been suggested that the transfer of L-glycero-alpha-D-manno-heptose (Hep) to Kdo by an E. coli WaaC may not be possible in the presence of (2 --> 8)-linked Kdo. E. coli double-mutants deficient in heptosyltransferases I (waaC) and II (waaF) and expressing waaA of Chlamydiae instead of their own, make Chlamydia-type Kdo oligosaccharides which are attached to an E. coli lipid A. Using such strains expressing waaA of Chlamydophila pneumoniae, Chlamydophila psittaci, or Chlamydia trachomatis, we have studied the effect of E. coli waaC gene expression on LPS structure. Structural analyses revealed the formation of two novel oligosaccharides Hep-(1 --> 5)[Kdo-(2 --> 4)]-Kdo and Hep-(1 --> 5)[Kdo-(2 --> 8)-Kdo-(2 --> 4)]-Kdo showing that Hep is transferred in the presence of (2 --> 8)-linked Kdo. Surprisingly, the transfer of Hep onto Kdo-(2 --> 4)-Kdo-(2 --> 4)-Kdo did not occur, despite the fact that Hep-(1 --> 5)[Kdo-(2 --> 4)-Kdo-(2 --> 4)]-Kdo is found in nature as a partial structure of E. coli LPS. The premature end of the biosynthesis and incorporation of Hep into the LPS indicated that WaaC had access to the substrate before Kdo transfer was completed. We have observed differences between WaaA of C. trachomatis, C. pneumoniae and C. psittaci which indicate mechanistic differences between these Kdo transferases.

Cloning, sequencing, and functional analysis of three glycosyltransferases involved in the biosynthesis of the inner core region of Klebsiella pneumoniae lipopolysaccharide

The genes encoding the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase (waaA) and heptosyltransferases I (waaC) and II (waaF) in Klebsiella pneumoniae were cloned from a DNA library by functional complementation of corresponding Escherichia coli and Salmonella enterica mutants. Sequence analyses revealed extensive homologies of the deduced proteins to their counterparts in other Enterobacteriaceae. However, differences were evident with regard to the chromosomal organization of the genes. To perform in vitro studies, the waaA, waaC and waaF genes were subcloned and expressed in the Gram-positive host Corynebacterium glutamicum. WaaA was characterized as a bifunctional enzyme capable of transferring two Kdo residues to a synthetic bisphosphorylated tetraacyl-lipid A precursor of E. coli (compound 406). In contrast, waaC and waaF were shown to encode specific glycosyltransferases catalyzing the consecutive transfer of two L-glycero-D-manno-heptose residues to Kdo(2)-406.