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 Role of Lipopolysaccharide Phase Variation in Susceptibility of Haemophilus influenzae to Bactericidal Immunoglobulin M Antibodies in Rabbit Sera

2000 ◽  
Vol 68 (5) ◽  
pp. 2804-2807 ◽  
Author(s):  
Alice L. Erwin ◽  
Yambasu A. Brewah ◽  
Debra A. Couchenour ◽  
Philip R. Barren ◽  
Stephen J. Burke ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Human Serum ◽  
Clinical Isolate ◽  
Bactericidal Activity ◽  
Phase Variation ◽  
Immunoglobulin M ◽  
Second Phase ◽  
Phase Variable ◽  
Igm Antibodies

ABSTRACT The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galα1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities ofH. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galα1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occuring bactericidal activity for unencapsulatedH. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

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.

Characterization of a dam mutant of Haemophilus influenzae Rd

Microbiology ◽  
2004 ◽  
Vol 150 (11) ◽  
pp. 3773-3781 ◽  
Author(s):  
Piotr Zaleski ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
High Sensitivity ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Phenotypic Properties ◽  
Chloramphenicol Resistance ◽  
Double Strand Dna Breaks ◽  
Dam Methylation ◽  
Dna Strands

The gene encoding Dam methyltransferase of Haemophilus influenzae was mutagenized by the insertion of a chloramphenicol-resistance cassette into the middle of the Dam coding sequence. This mutant construct was introduced into the H. influenzae chromosome by transformation and selection for CamR transformants. The authors have shown that several phenotypic properties, resistance to antibiotics, dyes and detergent as well as efficiency of transformation, depend on the Dam methylation state of the DNA. Although the major role of the methyl-directed mismatch repair (MMR) system is to repair postreplicative errors, it seems that in H. influenzae its effect is more apparent in repairing DNA damage caused by oxidative compounds. In the dam mutant treated with hydrogen peroxide, MMR is not targeted to newly replicated DNA strands and therefore mismatches are converted into single- and double-strand DNA breaks. This is shown by the increased peroxide sensitivity of the dam mutant and the finding that the sensitivity can be suppressed by a mutH mutation inactivating MMR. In the dam mutant treated with nitrofurazone the resulting damage is not converted into DNA breaks but the high sensitivity is also suppressed by a mutH mutation.

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 Role of the AcrAB-TolC Efflux Pump in Determining Susceptibility of Haemophilus influenzae to the Novel Peptide Deformylase Inhibitor LBM415

2005 ◽  
Vol 49 (8) ◽  
pp. 3129-3135 ◽  
Author(s):  
Charles R. Dean ◽  
Shubha Narayan ◽  
Denis M. Daigle ◽  
JoAnn L. Dzink-Fox ◽  
Xiaoling Puyang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Efflux Pump ◽  
Clinical Isolates ◽  
Peptide Deformylase ◽  
The Novel ◽  
Insertional Inactivation ◽  
Genetic Deletion ◽  
Increased Susceptibility

ABSTRACT Haemophilus influenzae isolates vary widely in their susceptibilities to the peptide deformylase inhibitor LBM415 (MIC range, 0.06 to 32 μg/ml); however, on average, they are less susceptible than gram-positive organisms, such as Staphylococcus aureus and Streptococcus pneumoniae. Insertional inactivation of the H. influenzae acrB or tolC gene in strain NB65044 (Rd strain KW20) increased susceptibility to LBM415, confirming a role for the AcrAB-TolC pump in determining resistance. Consistent with this, sequencing of a PCR fragment generated with primers flanking the acrRA region from an LBM415-hypersusceptible H. influenzae clinical isolate revealed a genetic deletion of acrA. Inactivation of acrB or tolC in several clinical isolates with atypically reduced susceptibility to LBM415 (MIC of 16 μg/ml or greater) significantly increased susceptibility, confirming that the pump is also a determinant of decreased susceptibility in these clinical isolates. Examination of acrR, encoding the putative repressor of pump gene expression, from several of these strains revealed mutations introducing frameshifts, stop codons, and amino acid changes relative to the published sequence, suggesting that loss of pump repression leads to decreased susceptibility. Supporting this, NB65044 acrR mutants selected by exposure to LBM415 at 8 μg/ml had susceptibilities to LBM415 and other pump substrates comparable to the least sensitive clinical isolates and showed increased expression of pump genes.

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 Identification of the Functional Initiation Codons of a Phase-Variable Gene of Haemophilus influenzae, lic2A, with the Potential for Differential Expression

2006 ◽  
Vol 189 (2) ◽  
pp. 511-521 ◽  
Author(s):  
Kevin Dixon ◽  
Christopher D. Bayliss ◽  
Katherine Makepeace ◽  
E. Richard Moxon ◽  
Derek W. Hood
Keyword(s):  
Gene Expression ◽  
Haemophilus Influenzae ◽  
Phase Variation ◽  
Site Directed Mutagenesis ◽  
Phase Variable ◽  
Tetranucleotide Repeat ◽  
Strong Expression ◽  
Variable Gene ◽  
Weak Expression ◽  
Reading Frames

ABSTRACT Simple sequence repeats located within reading frames mediate phase-variable ON/OFF switches in gene expression by generating frameshifts. Multiple translation initiation codons in different reading frames are found upstream of most Haemophilus influenzae tetranucleotide repeat tracts, raising the possibility of multiple active reading frames and more than two levels of gene expression for these loci. Phase variation between three levels of gene expression (strong, weak, and none) was observed when lic2A was fused to a lacZ reporter gene. The lic2A 5′ CAAT repeat tract is preceded by four 5′ ATG codons (x, y, z1, and z2) in two reading frames. Each of these initiation codons was inactivated by site-directed mutagenesis. Strong expression from frame 1 was associated with x but not y. Weak expression from frame 2 was mainly dependent on the z2 codon, and there was no expression from frame 3. Using monoclonal antibodies specific for a digalactoside epitope of lipopolysaccharide whose synthesis requires Lic2A, two levels (strong and undetectable) of antibody reactivity were detected, suggesting that weak expression of lic2A is not discernible at the phenotypic level. Inactivation of the x initiation codon resulted in loss of strong expression of the digalactoside epitope and elevated killing by human serum. The failure to detect more than two phenotypes for lic2A, despite clear evidence of weak expression from the z1/z2 initiation codons, leaves open the question of whether or not multiple initiation codons are associated with more complex patterns of phenotypic variation rather than classical phase-variable switching between two phenotypes.

scholarly journals Phase Variation in HMW1A Controls a Phenotypic Switch in Haemophilus influenzae Associated with Pathoadaptation during Persistent Infection

mBio ◽  
2021 ◽  
Author(s):  
Ariadna Fernández-Calvet ◽  
Begoña Euba ◽  
Celia Gil-Campillo ◽  
Arancha Catalan-Moreno ◽  
Javier Moleres ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Persistent Infection ◽  
Bacterial Pathogens ◽  
Phase Variation ◽  
Phase Variable ◽  
Phenotypic Switch

Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression.

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