Christopher D. Bayliss

Gene expression technology.

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The length of a tetranucleotide repeat tract in Haemophilus influenzae determines the phase variation rate of a gene with homology to type III DNA methyltransferases

Haemophilus influenzae is an obligate commensal of the upper respiratory tract of humans that uses simple repeats (microsatellites) to alter gene expression. The mod gene of H. influenzae strain Rd has homology to DNA methyltransferases of type III restriction/modification systems and has 40 tetranucleotide (5′‐AGTC) repeats within its open reading frame. This gene was found in 21 out of 23 genetically distinct H. influenzae strains, and in 13 of these strains the locus contained repeats. H. influenzae strains were constructed in which a lacZ reporter was fused to a chromosomal copy of mod downstream of the repeats. Phase variation occurred at a high frequency in strains with the wild‐type number of repeats. Mutation rates were derived for similarly engineered strains, containing different numbers of repeats. Rates increased linearly with tract length over the range 17–38 repeat units. The majority of tract alterations were insertions or deletions of one repeat unit with a 2:1 bias towards contractions of the tract. These results demonstrate the number of repeats to be an important determinant of phase variation rate in H. influenzae for a gene containing a microsatellite.

The simple sequence contingency loci of Haemophilus influenzae and Neisseria meningitidis

Many pathogens have evolved the ability to alter surface-exposed molecules, most often in response to selective pressures associated with the host immune system (1). Pathogenic bacteria exhibit numerous examples of this adaptive strategy, and a range of molecular mechanisms has evolved in these bacteria for generating genetic variation at individual loci termed “contingency loci” (2). Many contingency genes are controlled by simple sequence DNA repeats that accumulate reversible, rec-independent mutations at high frequency. A striking feature of the complete genome sequences of the human pathogens Haemophilus influenzae and Neisseria meningitidis is the abundance of loci containing simple sequences (3, 4). Intriguingly, both of these bacteria are obligate commensals of the upper respiratory tract of humans but can, in some hosts, cause life-threatening invasive disease. In this Perspective we provide a synthesis of our current understanding of how polymorphisms in simple sequence contingency loci produce phenotypic variation. We also evaluate the role of this variation in pathogenesis and the evolution of virulent bacterial strains.

Determinants of phase variation rate and the fitness implications of differing rates for bacterial pathogens and commensals

Phase variation (PV) of surface molecules and other phenotypes is a major adaptive strategy of pathogenic and commensal bacteria. Phase variants are produced at high frequencies and in a reversible manner by hypermutation or hypervariable methylation in specific regions of the genome. The major mechanisms of PV involve site-specific recombination, homologous recombination, simple sequence DNA repeat tracts or epigenetic modification by the dam methylase. PV rates of some of these mechanisms are subject to the influence of genome maintenance pathways such as DNA replication, recombination and repair while others are independent of these pathways. For each of these mechanisms, the rate of generation of phase variants is controlled by intrinsic and dispensable factors. These factors can impart environmental regulation on switching rates while many factors are subject to heterogeneity both within isolates of a species and between species. A major gap in our understanding is whether these environmental and epidemiological variations in PV rate have a major impact on fitness. Experimental approaches to studying the biological relevance of differing PV rates are being developed, and a recent intriguing finding is of a co-ordination of switching rates in the phase variable P-pili of uropathogenic bacteria.

Mutations in polI but not mutSLH destabilize Haemophilus influenzae tetranucleotide repeats.

Haemophilus influenzae (Hi), an obligate upper respiratory tract commensal/pathogen, uses phase variation (PV) to adapt to host environment changes. Switching occurs by slippage of nucleotide repeats (microsatellites) within genes coding for virulence molecules. Most such microsatellites in Hi are tetranucleotide repeats, but an exception is the dinucleotide repeats in the pilin locus. To investigate the effects on PV rates of mutations in genes for mismatch repair (MMR), insertion/deletion mutations of mutS, mutL, mutH, dam, polI, uvrD, mfd and recA were constructed in Hi strain Rd. Only inactivation of polI destabilized tetranucleotide (5′AGTC) repeat tracts of chromosomally located reporter constructs, whereas inactivation of mutS, but not polI, destabilized dinucleotide (5′AT) repeats. Deletions of repeats were predominant in polI mutants, which we propose are due to end‐joining occurring without DNA polymerization during polI‐deficient Okazaki fragment processing. The high prevalence of tetranucleotides mediating PV is an exceptional feature of the Hi genome. The refractoriness to MMR of hypermutation in Hi tetranucleotides facilitates adaptive switching without the deleterious increase in global mutation rates that accompanies a mutator genotype.

Persistence, Replacement, and Rapid Clonal Expansion of Meningococcal Carriage Isolates in a 2008 University Student Cohort

ABSTRACT A study of meningococcal carriage dynamics was performed with a cohort of 190 first-year students recruited from six residential halls at Nottingham University, United Kingdom. Pharyngeal swabs were obtained on four occasions between November 2008 and May 2009. Direct plating and culture on selective media were succeeded by identification and characterization of meningococci using PCR-based methodologies. Three serogroup Y clones and one serogroup 29E clone were highly prevalent in particular residential halls in November 2008, which is indicative of rapid clonal expansion since the start of the academic year. Persistent carriage of the same meningococcal strain for at least 5 to 6 months was observed in 45% of carriers, with infrequent evidence of antigenic variation in PorA. Sequential carriage of heterologous meningococcal strains occurred in 36% of carriers and involved strains with different capsules and antigenic variants of PorA and FetA in 83% of the cases. These clonal replacement strains also exhibited frequent differences in the presence and antigenic structures of two other surface proteins, NadA and HmbR. This study highlights the low level of antigenic variation associated with persistent carriage but, conversely, the importance of alterations in the repertoire of antigenic variants for sequential carriage of meningococcal strains. Rapid clonal expansion of potentially pathogenic strains in residential halls has implications for the implementation of public health interventions in university populations.

Influence of the combination and phase variation status of the haemoglobin receptors HmbR and HpuAB on meningococcal virulence.

Neisseria meningitidis can utilize haem, haemoglobin and haemoglobin–haptoglobin complexes as sources of iron via two TonB-dependent phase variable haemoglobin receptors, HmbR and HpuAB. HmbR is over-represented in disease isolates, suggesting a link between haemoglobin acquisition and meningococcal disease. This study compared the distribution of HpuAB and phase variation (PV) status of both receptors in disease and carriage isolates. Meningococcal disease (n = 214) and carriage (n = 305) isolates representative of multiple clonal complexes (CCs) were investigated for the distribution, polyG tract lengths and ON/OFF status of both haemoglobin receptors, and for the deletion mechanism for HpuAB. Strains with both receptors or only hmbR were present at similar frequencies among meningococcal disease isolates as compared with carriage isolates. However, >90 % of isolates from the three CCs CC5, CC8 and CC11 with the highest disease to carriage ratios contained both receptors. Strains with an hpuAB-only phenotype were under-represented among disease isolates, suggesting selection against this receptor during systemic disease, possibly due to the receptor having a high level of immunogenicity or being inefficient in acquisition of iron during systemic spread. Absence of hpuAB resulted from either complete deletion or replacement by an insertion element. In an examination of PV status, one or both receptors were found in an ON state in 91 % of disease and 71 % of carriage isolates. We suggest that expression of a haemoglobin receptor, either HmbR or HpuAB, is of major importance for systemic spread of meningococci, and that the presence of both receptors contributes to virulence in some strains.

Neisseria meningitidis escape from the bactericidal activity of a monoclonal antibody is mediated by phase variation of lgtG and enhanced by a mutator phenotype.

ABSTRACT Bacteria adapt to environmental changes through high-frequency switches in expression of specific phenotypes. Localized hypermutation mediated by simple sequence repeats is an important mechanism of such phase variation (PV) in Neisseria meningitidis. Loss or gain of nucleotides in a poly(C) tract located in the reading frame results in switches in expression of lgtG and determines whether a glucose or a phosphoethanolamine (PEtn) is added at a specific position in the inner core lipopolysaccharide (LPS). Monoclonal antibody (MAb) B5 is bactericidal for N. meningitidis strain 8047 when PEtn is present in the inner core LPS and lgtG is switched “off.” Escape from the bactericidal activity of this antibody was examined by subjecting strain 8047 to multiple cycles of growth in the presence of MAb B5 and human serum. Escape variants with alterations in the lgtG repeat tract rapidly accumulated in bacterial populations during selection with this antibody. Strain 8047 was outcompeted in this assay by the 8047 ΔmutS strain due to the elevated PV rate of this mismatch repair mutant and hence the greater proportion of preexisting phase variants of lgtG in the inoculum. This mutS mutant was also more virulent than strain 8047 during escape from passive protection by MAb B5 in an in vivo infant rat model of bacteremia. These results provide an example of how PV rates can modulate the occurrence and severity of infection and have important implications for understanding the evolution of bacterial fitness in species subject to environmental variations that occur during persistence within and transmission between hosts.

Carriage of meningococci by university students, United Kingdom.

To the Editor: Neisseria meningitidis causes septicemia and meningitis (1). Meningococci usually persist on the nasopharyngeal mucosa of asymptomatic carriers (2). Because carriers are the only reservoir of meningococci, carriage in at-risk populations should be monitored. Meningococcal carriage rates have been assessed during 1997–8 for first-year students at the University of Nottingham (3) and in autumn during 1999–2001 for >48,000 sixth-form students (pre-university, age range 15–17 years) throughout the United Kingdom (4). Serogroup B and nongroupable strains predominated; serogroup Y strains were found in only 1%–2% of participants. From November 2008 through May 2009, to investigate persistence and spread of meningococcal strains in students living in dormitories, we conducted a longitudinal study in a cohort of 190 first-year students at the University of Nottingham. We found high rates of carriage and prevalence of serogroup Y strains (5). During September 2009 (first week of term) through March 2010, we conducted a large repeated cross-sectional study analyzing pharyngeal swabs from students in all school-year groups at Nottingham University. The objective of this study was to determine the significance of changes in overall meningococcal and serogroup Y-specific carriage rates among students. In September, first-year students were recruited on the main campus during registration and subsequently in dormitories and the main library. Undergraduates not in the first year were all recruited in the main library. This September sample of 823 first-year students represents 16.5% of the 5,000 undergraduate students registered each academic year on the main campus. Although not intentional, some overlap occurred when students were resampled during subsequent visits to the same dormitories and library, e.g., among the 557 first-year students from whom swab samples were collected in December, 74 (13%) had previously provided swab samples. Our study was approved by the Nottingham University Medical School Ethics Committee, and written informed consent was obtained from all participants. Pharyngeal swab samples were spread onto GC selective agar (Oxoid, Basingstoke, UK) and incubated at 37°C in air containing 5% CO2. After 48 hours, colonies suggestive of Neisseria spp. were examined for positive oxidase reaction; single colonies were confirmed as meningococci by amplification of meningococcal genes crgA plus ctrA and/or porA (6). PCR-based serogrouping was performed as described (6,7). Chi-square tests for significance were performed by using STATCALC (Epi Info version 6.04; Centers for Disease Control and Prevention, Atlanta, GA, USA). Among first-year students, carriage rates increased from 23.2% in late September to 55.7% by mid-December and remained at a similar level in March (Table). Among second- and third-year students, carriage rates were 34.2% and 30.5% in September, respectively, and remained at similar levels throughout the academic year. The increase in carriage among first-year students from September through December was mainly the result of a significant (23%) increase in carriage of serogroup Y strains (Table). In contrast, during the same period, carriage rates of serogroup Y strains did not change significantly among second- and third-year students (Table). Table Characteristics of meningococci carriage, University of Nottingham students, United Kingdom, 2009–10* Initial carriage rates were significantly higher for incoming (first-year) students in September 2009 than in 1997 (13.9% [3]; χ2 = 14, 1 df; p<0.0001); swabbing and culture protocols and sampling sites were identical in both studies, so the increases are real. Because 83% of students at Nottingham University come from all regions of the United Kingdom and 17% from other countries, the increased rates of carriage may reflect a nationwide change (8). Furthermore, testing within the first week of term meant that recovered strains were predominately brought into the university. Serogroup Y carriage rates for incoming students (2.9%) were significantly higher than rates detected by identical genotyping methods during 1999–2001 (1.7%–1.8% [4]; χ2 = 4.6%–6.4%, 1 df; p<0.05), suggesting that meningococcal carriage by young adults, particularly of serogroup Y strains, has increased across the United Kingdom. The major increase in serogroup Y strains among first-year students during 2009–10 probably resulted from spread of clones within dormitories, as observed in the 2008–9 study (5) and may be facilitated by characteristics of the organism, lack of immunity, or a combination of these factors. The high prevalence of serogroup Y strains in carriers may help explain the recent increased incidence of serogroup Y disease in the United Kingdom: from 20 to 62 laboratory-confirmed cases in England and Wales from 2003 through 2009 (9). In the United States during the late 1990s, a similar increase in serogroup Y carriage was linked to a concomitant increase in serogroup Y disease (10). In conclusion, in a representative UK student cohort we detected high rates of carriage and elevated prevalence of serogroup Y strains of meningococci. Any further significant increase in serogroup Y disease should lead to prompt reconsideration of the current vaccine policy in the United Kingdom.

High allelic diversity in the methyltransferase gene of a phase variable type III restriction-modification system has implications for the fitness of Haemophilus influenzae

Phase variable restriction-modification (R-M) systems are widespread in Eubacteria. Haemophilus influenzae encodes a phase variable homolog of Type III R-M systems. Sequence analysis of this system in 22 non-typeable H.influenzae isolates revealed a hypervariable region in the central portion of the mod gene whereas the res gene was conserved. Maximum likelihood (ML) analysis indicated that most sites outside this hypervariable region experienced strong negative selection but evidence of positive selection for a few sites in adjacent regions. A phylogenetic analysis of 61 Type III mod genes revealed clustering of these H.influenzae mod alleles with mod genes from pathogenic Neisseriae and, based on sequence analysis, horizontal transfer of the mod–res complex between these species. Neisserial mod alleles also contained a hypervariable region and all mod alleles exhibited variability in the repeat tract. We propose that this hypervariable region encodes the target recognition domain (TRD) of the Mod protein and that variability results in alterations to the recognition sequence of this R-M system. We argue that the high allelic diversity and phase variable nature of this R-M system have arisen due to selective pressures exerted by diversity in bacteriophage populations but also have implications for other fitness attributes of these bacterial species.