scholarly journals Mutations in Haemophilus influenzae Mismatch Repair Genes Increase Mutation Rates of Dinucleotide Repeat Tracts but Not Dinucleotide Repeat-Driven Pilin Phase Variation Rates

2004 ◽  
Vol 186 (10) ◽  
pp. 2928-2935 ◽  
Author(s):  
Christopher D. Bayliss ◽  
Wendy A. Sweetman ◽  
E. Richard Moxon
Keyword(s):  
Haemophilus Influenzae ◽  
Mismatch Repair ◽  
Bacterial Species ◽  
Phase Variation ◽  
Dinucleotide Repeat ◽  
Phase Variable ◽  
Upper Respiratory Tract ◽  
E Coli ◽  
Serotype B ◽  
Repair Genes

ABSTRACT High-frequency, reversible switches in expression of surface antigens, referred to as phase variation (PV), are characteristic of Haemophilus influenzae. PV enables this bacterial species, an obligate commensal and pathogen of the human upper respiratory tract, to adapt to changes in the host environment. Phase-variable hemagglutinating pili are expressed by many H. influenzae isolates. PV involves alterations in the number of 5′ TA repeats located between the −10 and −35 promoter elements of the overlapping, divergently orientated promoters of hifA and hifBCDE, whose products mediate biosynthesis and assembly of pili. Dinucleotide repeat tracts are destabilized by mismatch repair (MMR) mutations in Escherichia coli. The influence of mutations in MMR genes of H. influenzae strain Rd on dinucleotide repeat-mediated PV rates was investigated by using reporter constructs containing 20 5′ AT repeats. Mutations in mutS, mutL, and mutH elevated rates approximately 30-fold, while rates in dam and uvrD mutants were increased 14- and 3-fold, respectively. PV rates of constructs containing 10 to 12 5′ AT repeats were significantly elevated in mutS mutants of H. influenzae strains Rd and Eagan. An intact hif locus was found in 14 and 12% of representative nontypeable H. influenzae isolates associated with either otitis media or carriage, respectively. Nine or more tandem 5′ TA repeats were present in the promoter region. Surprisingly, inactivation of mutS in two serotype b H. influenzae strains did not alter pilin PV rates. Thus, although functionally analogous to the E. coli MMR pathway and active on dinucleotide repeat tracts, defects in H. influenzae MMR do not affect 5′ TA-mediated pilin PV.

scholarly journals Elucidation of the Monoclonal Antibody 5G8-Reactive, Virulence-Associated Lipopolysaccharide Epitope of Haemophilus influenzae and Its Role in Bacterial Resistance to Complement-Mediated Killing

2005 ◽  
Vol 73 (4) ◽  
pp. 2213-2221 ◽  
Author(s):  
Ruth Griffin ◽  
Andrew D. Cox ◽  
Katherine Makepeace ◽  
James C. Richards ◽  
E. Richard Moxon ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Haemophilus Influenzae ◽  
Bacterial Resistance ◽  
Inner Core ◽  
Phase Variation ◽  
Phase Variable ◽  
Type B ◽  
Variable Expression ◽  
Virulent Type ◽  
Unknown Structure

ABSTRACT The phase-variable locus lex2 is required for expression of a Haemophilus influenzae lipopolysaccharide (LPS) epitope of previously unknown structure. This epitope, which is reactive with monoclonal antibody (MAb) 5G8, has been associated with virulence of type b strains. When strain RM118 (from the same source as strain Rd), in which the lex2 locus and MAb 5G8 reactivity are absent, was transformed with lex2 DNA, transformants that were reactive with MAb 5G8 were obtained. Surprisingly, the 5G8 reactivity of these transformants was phase variable, although the lex2 locus lacked tetrameric repeats and was constitutively expressed. This phase variation was shown to be the result of phase-variable expression of phosphorylcholine (PCho) such that MAb 5G8 reacted only in the absence of PCho. Structural analysis showed that, compared to RM118, the lex2 transformant had acquired a tetrasaccharide, Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β1,4, linked to the proximal heptose (HepI). A terminal GalNAc was detected in a minority of glycoforms. LPS derived from a mutant of RM7004, a virulent type b strain which naturally expresses lex2 and has LPS containing the same tetrasaccharide linked to HepI as the sole oligosaccharide extension from the inner core, confirmed that GalNAc is not a part of the MAb 5G8-reactive epitope. Thus, MAb 5G8 specifically binds to the structure Gal-α1,4-Gal-β1,4-Glc-β1,4-Glc-β attached via a 1,4 linkage to HepI of H. influenzae LPS, and we show that the ability to synthesize this novel tetrasaccharide was associated with enhanced bacterial resistance to complement-mediated killing.

scholarly journals Redox Signal Transduction by the ArcB Sensor Kinase of Haemophilus influenzae Lacking the PAS Domain

2001 ◽  
Vol 183 (24) ◽  
pp. 7206-7212 ◽  
Author(s):  
Dimitris Georgellis ◽  
Ohsuk Kwon ◽  
Edmund C. C. Lin ◽  
Sandy M. Wong ◽  
Brian J. Akerley
Keyword(s):  
Signal Transduction ◽  
Haemophilus Influenzae ◽  
Bacterial Species ◽  
Redox Conditions ◽  
Sensor Kinase ◽  
Pas Domain ◽  
Signal Transduction System ◽  
E Coli ◽  
Two Component

ABSTRACT The Arc (anoxic redox control) two-component signal transduction system of Escherichia coli, which comprises the tripartite ArcB sensor kinase and the ArcA response regulator, modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the arcA and arcBgenes of Haemophilus influenzae specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro. Moreover, the Arc system of H. influenzae mediates transcriptional control according to the redox condition of growth both autologously in its own host and homologously in E. coli, indicating a high degree of functional conservation of the signal transduction system. The H. influenzae ArcB, however, lacks the PAS domain present in the region of E. coli ArcB linking the transmembrane to the cytosolic catalytic domains. Because the PAS domain participates in signal reception in a variety of sensory proteins, including sensors of molecular oxygen and redox state, a similar role was previously ascribed to it in ArcB. Our results demonstrate that the ArcB protein of H. influenzae mediates signal transduction in response to redox conditions of growth despite the absence of the PAS domain.

scholarly journals The role of Dam methylation in phase variation of Haemophilus influenzae genes involved in defence against phage infection

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3361-3369 ◽  
Author(s):  
Piotr Zaleski ◽  
Marek Wojciechowski ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Phage Infection ◽  
Phase Variable ◽  
Repeat Tract ◽  
Insertional Inactivation ◽  
Phage Dna ◽  
Dam Methylation ◽  
Tetranucleotide Repeats

Haemophilus influenzae uses phase variation (PV) to modulate the activity of its defence systems against phage infection. The PV of the restriction–modification (R-M) system HindI, the main defence system against phage infection and incoming chromosomal and phage DNA in H. influenzae Rd, is driven by changes of the pentanucleotide repeat tract within the coding sequence of the hsdM gene and is influenced by lack of Dam methylation. Phase-variable resistance/sensitivity to phage infection correlates with changes in lipooligosaccharide (LOS) structure and occurs by slippage of tetranucleotide repeats within the gene lic2A, coding for a step in the biosynthesis of LOS. The lack of Dam activity destabilizes the tetranuclotide (5′-CAAT) repeat tract and increases the frequency of switching from sensitivity to resistance to phage infection more than in the opposite direction. The PV of the lgtC gene does not influence resistance or sensitivity to phage infection. Insertional inactivation of lic2A, but not lgtC or lgtF, leads to resistance to phage infection and to the same structure of the LOS as observed among phase-variable phage-resistant variants. This indicates that in the H. influenzae Rd LOS only the first two sugars (Glc-Gal) extending from the third heptose are part of bacterial phage receptors.

scholarly journals VapC-1 of Nontypeable Haemophilus influenzae Is a Ribonuclease

2007 ◽  
Vol 189 (14) ◽  
pp. 5041-5048 ◽  
Author(s):  
Dayle A. Daines ◽  
Mack H. Wu ◽  
Sarah Y. Yuan
Keyword(s):  
Haemophilus Influenzae ◽  
Growth Inhibition ◽  
Gene Pair ◽  
Gene Families ◽  
Upper Respiratory Tract ◽  
Nontypeable Haemophilus Influenzae ◽  
E Coli ◽  
Conserved Gene

ABSTRACT Nontypeable Haemophilus influenzae (NTHi) organisms are obligate parasites of the human upper respiratory tract that can exist as commensals or pathogens. Toxin-antitoxin (TA) loci are highly conserved gene pairs that encode both a toxin and antitoxin moiety. Seven TA gene families have been identified to date, and NTHi carries two alleles of the vapBC family. Here, we have characterized the function of one of the NTHi alleles, vapBC-1. The gene pair is transcribed as an operon in two NTHi clinical isolates, and promoter fusions display an inverse relationship to culture density. The antitoxin VapB-1 forms homomultimers both in vitro and in vivo. The expression of the toxin VapC-1 conferred growth inhibition to an Escherichia coli expression strain and was successfully purified only when cloned in tandem with its cognate antitoxin. Using total RNA isolated from both E. coli and NTHi, we show for the first time that VapC-1 is an RNase that is active on free RNA but does not degrade DNA in vitro. Preincubation of the purified toxin and antitoxin together results in the formation of a protein complex that abrogates the activity of the toxin. We conclude that the NTHi vapBC-1 gene pair functions as a classical TA locus and that the induction of VapC-1 RNase activity leads to growth inhibition via the mechanism of mRNA cleavage.

Induction of the SOS regulon of Haemophilus influenzae does not affect phase variation rates at tetranucleotide or dinucleotide repeats

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2751-2763 ◽  
Author(s):  
Wendy A. Sweetman ◽  
E. Richard Moxon ◽  
Christopher D. Bayliss
Keyword(s):  
Haemophilus Influenzae ◽  
Uv Irradiation ◽  
Phase Variation ◽  
Sos Response ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Dinucleotide Repeats ◽  
Tetranucleotide Repeat ◽  
E Coli ◽  
Lexa Binding

Haemophilus influenzae has microsatellite repeat tracts in 5′ coding regions or promoters of several genes that are important for commensal and virulence behaviour. Changes in repeat number lead to switches in expression of these genes, a process referred to as phase variation. Hence, the virulence behaviour of this organism may be influenced by factors that alter the frequency of mutations in these repeat tracts. In Escherichia coli, induction of the SOS response destabilizes dinucleotide repeat tracts. H. influenzae encodes a homologue of the E. coli SOS repressor, LexA. The H. influenzae genome sequence was screened for the presence of the minimal consensus LexA-binding sequence from E. coli, CTG(N)10CAG, in order to identify genes with the potential to be SOS regulated. Twenty-five genes were identified that had LexA-binding sequences within 200 bp of the start codon. An H. influenzae non-inducible LexA mutant (lexA NI) was generated by site-directed mutagenesis. This mutant showed increased sensitivity, compared with wild-type (WT) cells, to both UV irradiation and mitomycin C (mitC) treatment. Semi-quantitative RT-PCR studies confirmed that H. influenzae mounts a LexA-regulated SOS response following DNA assault. Transcript levels of lexA, recA, recN, recX, ruvA and impA were increased in WT cells following DNA damage but not in lexA NI cells. Induction of the H. influenzae SOS response by UV irradiation or mitC treatment did not lead to any observable SOS-dependent changes in phase variation rates at either dinucleotide or tetranucleotide repeat tracts. Treatment with mitC caused a small increase in phase variation rates in both repeat tracts, independently of an SOS response. We suggest that the difference between H. influenzae and E. coli with regard to the effect of the SOS response on dinucleotide phase variation rates is due to the absence of any of the known trans-lesion synthesis DNA polymerases in H. influenzae.

scholarly journals Genotypic and Phenotypic Characterization of theO-Linked Protein Glycosylation System Reveals High Glycan Diversity in Paired Meningococcal Carriage Isolates

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Bente Børud ◽  
Guro K. Bårnes ◽  
Ola Brønstad Brynildsrud ◽  
Elisabeth Fritzsønn ◽  
Dominique A. Caugant
Keyword(s):  
Neisseria Meningitidis ◽  
Phase Variation ◽  
Protein Glycosylation ◽  
Phase Variable ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Variable Expression ◽  
Evolutionary Forces ◽  
Strain Collection

ABSTRACTSpecies within the genusNeisseriadisplay significant glycan diversity associated with theO-linked protein glycosylation (pgl) systems due to phase variation and polymorphic genes and gene content. The aim of this study was to examine in detail thepglgenotype and glycosylation phenotype in meningococcal isolates and the changes occurring during short-term asymptomatic carriage. Paired meningococcal isolates derived from 50 asymptomatic meningococcal carriers, taken about 2 months apart, were analyzed with whole-genome sequencing. TheO-linked protein glycosylation genes were characterized in detail using the Genome Comparator tool at the https://pubmlst.org/ database. Immunoblotting with glycan-specific antibodies (Abs) was used to investigate the protein glycosylation phenotype. All majorpgllocus polymorphisms identified inNeisseria meningitidisto date were present in our isolate collection, with the variable presence ofpglGandpglH, both in combination with eitherpglBorpglB2. We identified significant changes and diversity in thepglgenotype and/or glycan phenotype in 96% of the paired isolates. There was also a high degree of glycan microheterogeneity, in which different variants of glycan structures were found at a given glycoprotein. The main mechanism responsible for the observed differences was phase-variable expression of the involved glycosyltransferases and theO-acetyltransferase. To our knowledge, this is the first characterization of thepglgenotype and glycosylation phenotype in a larger strain collection. This report thus provides important insight into glycan diversity inN. meningitidisand into the phase variability changes that influence the expressed glycoform repertoire during meningococcal carriage.IMPORTANCEBacterial meningitis is a serious global health problem, and one of the major causative organisms isNeisseria meningitidis, which is also a common commensal in the upper respiratory tract of healthy humans. In bacteria, numerous loci involved in biosynthesis of surface-exposed antigenic structures that are involved in the interaction between bacteria and host are frequently subjected to homologous recombination and phase variation. These mechanisms are well described inNeisseria, and phase variation provides the ability to change these structures reversibly in response to the environment. Protein glycosylation systems are becoming widely identified in bacteria, and yet little is known about the mechanisms and evolutionary forces influencing glycan composition during carriage and disease.

scholarly journals Phase Variation During Host Colonization and Invasion by Campylobacter jejuni and Other Campylobacter Species

2021 ◽  
Vol 12 ◽  
Author(s):  
Caroline Cayrou ◽  
Natalie A. Barratt ◽  
Julian M. Ketley ◽  
Christopher D. Bayliss
Keyword(s):  
Bacterial Species ◽  
Foodborne Pathogen ◽  
Phase Variation ◽  
Phase Variable ◽  
Bacterial Survival ◽  
Zoonotic Infections ◽  
Infection Models ◽  
The Status ◽  
Host Tissues ◽  
Variable Genes

Phase variation (PV) is a phenomenon common to a variety of bacterial species for niche adaption and survival in challenging environments. Among Campylobacter species, PV depends on the presence of intergenic and intragenic hypermutable G/C homopolymeric tracts. The presence of phase-variable genes is of especial interest for species that cause foodborne or zoonotic infections in humans. PV influences the formation and the structure of the lipooligosaccharide, flagella, and capsule in Campylobacter species. PV of components of these molecules is potentially important during invasion of host tissues, spread within hosts and transmission between hosts. Motility is a critical phenotype that is potentially modulated by PV. Variation in the status of the phase-variable genes has been observed to occur during colonization in chickens and mouse infection models. Interestingly, PV is also involved in bacterial survival of attack by bacteriophages even during chicken colonization. This review aims to explore and discuss observations of PV during model and natural infections by Campylobacter species and how PV may affect strategies for fighting infections by this foodborne pathogen.

scholarly journals Lex2B, a Phase-Variable Glycosyltransferase, Adds either a Glucose or a Galactose to Haemophilus influenzae Lipopolysaccharide

2009 ◽  
Vol 77 (6) ◽  
pp. 2376-2384 ◽  
Author(s):  
M. E. Deadman ◽  
P. Hermant ◽  
M. Engskog ◽  
K. Makepeace ◽  
E. R. Moxon ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Respiratory Tract Infections ◽  
Inner Core ◽  
Biological Properties ◽  
Single Amino Acid ◽  
Phase Variable ◽  
Killing Effect ◽  
Variable Expression ◽  
Serotype B ◽  
Tract Infections

ABSTRACT Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second β-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.

scholarly journals Broad Conditions Favor the Evolution of Phase-Variable Loci

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
M. E. Palmer ◽  
M. Lipsitch ◽  
E. R. Moxon ◽  
C. D. Bayliss
Keyword(s):  
Haemophilus Influenzae ◽  
In Silico ◽  
Simple Sequence Repeat ◽  
Phase Variation ◽  
Phase Variable ◽  
Infection Cycle ◽  
Sequence Repeat ◽  
Ssr Loci ◽  
Stochastic Switching ◽  
Simple Sequence

ABSTRACT Simple sequence repeat (SSR) tracts produce stochastic on-off switching, or phase variation, in the expression of a panoply of surface molecules in many bacterial commensals and pathogens. A change to the number of repeats in a tract may alter the phase of the translational reading frame, which toggles the on-off state of the switch. Here, we construct an in silico SSR locus with mutational dynamics calibrated to those of the Haemophilus influenzae mod locus. We simulate its evolution in a regimen of two alternating environments, simultaneously varying the selection coefficient, s, and the epoch length, T. Some recent work in a simpler (two-locus) model suggested that stochastic switching in a regimen of two alternating environments may be evolutionarily favored only if the selection coefficients in the two environments are nearly equal (“symmetric”) or selection is very strong. This finding was puzzling, as it greatly restricted the conditions under which stochastic switching might evolve. Instead, we find agreement with other recent theoretical work, observing selective utility for stochastic switching if the product sT is large enough for the favored state to nearly fix in both environments. Symmetry is required neither in s nor in sT. Because we simulate finite populations and use a detailed model of the SSR locus, we are also able to examine the impact of population size and of several SSR locus parameters. Our results indicate that conditions favoring evolution and maintenance of SSR loci in bacteria are quite broad. IMPORTANCE Bacteria experience frequent changes of environment during the infection cycle. One means to rapidly adapt is stochastic switching: a bacterial lineage will stochastically produce a variety of genotypes, so that some descendants will survive if the environment changes. Stochastic switching mediated by simple sequence repeat (SSR) loci is widespread among bacterial commensals and pathogens and influences critical interactions with host surfaces or immune effectors, thereby affecting host persistence, transmission, and virulence. Here, we use the most detailed in silico model of an SSR locus to date, with its phase variation calibrated to match the mod locus of Haemophilus influenzae. The type III restriction-modification system encoded by mod participates in the regulation of multiple other genes; thus, SSR-mediated phase variation of mod has far-reaching cis-regulatory effects. This coupling of phase-variable switching to complex phenotypic effects has been described as the “phasevarion” and is central to understanding the infection cycle of bacterial commensals and pathogens.

scholarly journals Molecular Cloning of the fur Gene from Actinobacillus actinomycetemcomitans

2002 ◽  
Vol 70 (6) ◽  
pp. 3170-3179 ◽  
Author(s):  
V. I. Haraszthy ◽  
E. T. Lally ◽  
G. G. Haraszthy ◽  
J. J. Zambon
Keyword(s):  
Gene Product ◽  
Bacterial Species ◽  
Consensus Sequence ◽  
Actinobacillus Actinomycetemcomitans ◽  
Gene Probe ◽  
Virulence Determinants ◽  
Chromosomal Dna ◽  
E Coli ◽  
Therapeutic Modalities ◽  
Serotype B

ABSTRACT In several bacterial species, iron availability in host tissues is coordinated with the expression of virulence determinants through the fur gene product. Initial experiments showed that a cloned Escherichia coli fur gene probe hybridized to Southern blots of Actinobacillus actinomycetemcomitans strain JP2 (serotype b) chromosomal DNA. The A. actinomycetemcomitans fur gene was then cloned utilizing partial functional complementation of the fur mutant in E. coli strain H1780. Analysis of the cloned DNA sequence revealed a 438-bp open reading frame with a deduced 146-amino-acid sequence exhibiting 80% identity to Haemophilus influenzae Fur and 62% identity to E. coli Fur. The pUC Aafur gene probe (generated from JP2 serotype b) hybridized to representatives from all five A. actinomycetemcomitans serotypes as well as to two strains derived from monkeys, suggesting that fur is widely distributed in A. actinomycetemcomitans. Open reading frames having >70% identity with the E. coli and H. influenzae flavodoxin and gyrase A genes, respectively, were found. Expression of the A. actinomycetemcomitans fur gene product repressed fiu expression and siderophore production in E. coli. A gel shift assay demonstrated that the expressed A. actinomycetemcomitans Fur protein bound the bacterial fur consensus sequence. Further characterization of the fur gene product in A. actinomycetemcomitans may improve our understanding of its role in the pathogenesis of periodontal disease and may lead to specific therapeutic modalities.

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