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.

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 Phase variable glycosylation in non-typeable Haemophilus influenzae

2019 ◽  
Author(s):  
Danila Elango ◽  
Benjamin L. Schulz
Keyword(s):  
Haemophilus Influenzae ◽  
Ex Vivo ◽  
Phase Variation ◽  
Population Diversity ◽  
Site Directed Mutagenesis ◽  
Host Cells ◽  
Phase Variable ◽  
Repeat Elements ◽  
C Terminus ◽  
Tract Infections

AbstractNon-typeable Haemophilus influenzae (NTHi) is a leading cause of respiratory tract infections worldwide and continues to be a global health burden. Adhesion and colonisation of host cells are crucial steps in bacterial pathogenesis, and in many strains of NTHi interaction with the host is mediated by the high molecular weight adhesins HMW1A and HMW2A. These adhesins are N-glycoproteins which are modified by cytoplasmic glycosyltransferases HMW1C and HMW2C. Phase variation in the number of short sequence repeats in the promoters of hmw1A and hmw2A directly affects their expression. Here, we report the presence of similar variable repeat elements in the promoters of hmw1C and hmw2C in diverse NTHi isolates. In an ex vivo assay, we systematically altered substrate and glycosyltransferase expression and showed that both of these factors affected the site-specific efficiency of glycosylation on HMW-A. Glycosylation occupancy was incomplete at many sites, variable between sites, and generally lower close to the C-terminus of HMW-A. We investigated the causes of this variability. As HMW-C glycosylates HMW-A in the cytoplasm, we tested how secretion affected glycosylation on HMW-A and showed that retaining HMW-A in the cytoplasm indeed increased glycosylation occupancy across the full length of the protein. Site-directed mutagenesis showed that HMW-C had no inherent preference for glycosylating asparagines in NxS or NxT sequons. This work provides key insights into factors contributing to the heterogenous modifications of NTHi HMW-A adhesins, expands knowledge of NTHi population diversity and pathogenic capability, and is relevant to vaccine design for NTHi and related pathogens.

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 Analysis of Invasive NontypeableHaemophilus influenzaeIsolates Reveals Selection for the Expression State of Particular Phase-Variable Lipooligosaccharide Biosynthetic Genes

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Zachary N. Phillips ◽  
Charles Brizuela ◽  
Amy V. Jennison ◽  
Megan Staples ◽  
Keith Grimwood ◽  
...  
Keyword(s):  
Gene Expression ◽  
Phase Variation ◽  
Phase Variable ◽  
Chronic Infections ◽  
Biosynthetic Genes ◽  
Content Type ◽  
Invasive Infections ◽  
Acetyl Group ◽  
Overt Disease ◽  
Expression Status

ABSTRACTNontypeableHaemophilus influenzae(NTHi) is a major human pathogen, responsible for several acute and chronic infections of the respiratory tract. The incidence of invasive infections caused by NTHi is increasing worldwide. NTHi is able to colonize the nasopharynx asymptomatically, and the exact change(s) responsible for transition from benign carriage to overt disease is not understood. We have previously reported that phase variation (the rapid and reversible ON-OFF switching of gene expression) of particular lipooligosaccharide (LOS) glycosyltransferases occurs during transition from colonizing the nasopharynx to invading the middle ear. Variation in the structure of the LOS is dependent on the ON/OFF expression status of each of the glycosyltransferases responsible for LOS biosynthesis. In this study, we surveyed a collection of invasive NTHi isolates for ON/OFF expression status of seven phase-variable LOS glycosyltransferases. We report that the expression state of the LOS biosynthetic genesoafAON andlic2AOFF shows a correlation with invasive NTHi isolates. We hypothesize that these gene expression changes contribute to the invasive potential of NTHi. OafA expression, which is responsible for the addition of anO-acetyl group onto the LOS, has been shown to impart a phenotype of increased serum resistance and may serve as a marker for invasive NTHi.

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 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 Transcriptome Sequencing Data Sets for Determining Gene Expression Changes Mediated by Phase-Variable DNA Methyltransferases in Nontypeable Haemophilus influenzae Strains Isolated from Patients with Chronic Obstructive Pulmonary Disease

2019 ◽  
Vol 8 (29) ◽  
Author(s):  
John M. Atack ◽  
Timothy F. Murphy ◽  
Melinda M. Pettigrew ◽  
Kate L. Seib ◽  
Michael P. Jennings
Keyword(s):  
Gene Expression ◽  
Chronic Obstructive Pulmonary Disease ◽  
Haemophilus Influenzae ◽  
Pulmonary Disease ◽  
Transcriptome Sequencing ◽  
Dna Methyltransferases ◽  
Chronic Obstructive ◽  
Phase Variable ◽  
Sequencing Data ◽  
Obstructive Pulmonary Disease

Nontypeable Haemophilus influenzae (NTHi) is a major bacterial cause of exacerbations in chronic obstructive pulmonary disease (COPD). Here, we report high-depth coverage transcriptome sequencing (RNA-seq) data from two NTHi strains, each encoding a different phase-variable methyltransferase. modA phase variation results in gene expression differences. These data will serve as an important resource for future studies.

scholarly journals Global Changes in Mycoplasma gallisepticum Phase-Variable Lipoprotein GenevlhAExpression duringIn VivoInfection of the Natural Chicken Host

2015 ◽  
Vol 84 (1) ◽  
pp. 351-355 ◽  
Author(s):  
K. Pflaum ◽  
E. R. Tulman ◽  
J. Beaudet ◽  
X. Liao ◽  
S. J. Geary
Keyword(s):  
Gene Expression ◽  
Phase Variation ◽  
Mycoplasma Gallisepticum ◽  
Chronic Respiratory Disease ◽  
Etiologic Agent ◽  
Economic Losses ◽  
Global Changes ◽  
Phase Variable ◽  
Content Type ◽  
Host Interaction

Mycoplasma gallisepticumis the primary etiologic agent of chronic respiratory disease in poultry, a disease largely affecting the respiratory tract and causing significant economic losses worldwide. Immunodominant proteins encoded by members of the variable lipoprotein and hemagglutinin (vlhA) gene family are thought to be important for mechanisms ofM. gallisepticum-host interaction, pathogenesis, and immune evasion, but their exact role and the overall nature of their phase variation are unknown. To better understand these mechanisms, we assessed global transcriptomicvlhAgene expression directly fromM. gallisepticumpopulations present on tracheal mucosae during a 7-day experimental infection in the natural chicken host. Here we report differences in both dominant and minorvlhAgene expression levels throughout the first week of infection and starting as early as day 1 postinfection, consistent with a functional role not dependent on adaptive immunity for driving phase variation. Notably, data indicated that, at given time points, specificvlhAgenes were similarly dominant in multiple independent hosts, suggesting a nonstochastic temporal progression of dominantvlhAgene expression in the colonizing bacterial population. The dominant expression of a givenvlhAgene was not dependent on the presence of 12-copy GAA trinucleotide repeats in the promoter region and did not revert to the predominatevlhAgene when no longer faced with host pressures. Overall, these data indicate thatvlhAphase variation is dynamic throughout the earliest stages of infection and that the pattern of dominantvlhAexpression may be nonrandom and regulated by previously unrecognized mechanisms.

Sign in / Sign up

Export Citation Format

Share Document