Error bars represent standard error of the mean titer of triplicate measurements in three independent experiments

Error bars represent standard error of the mean titer of triplicate measurements in three independent experiments. point mutations in the capsule synthesis genes substantially contribute to the repertoire of genetic mechanisms in natural populations leading Mouse monoclonal to RAG2 to variability in capsule expression. KEYWORDS:Neisseria meningitidis, capsule synthesis locus, genomic diversity, herd immunity, virulence == Introduction == Neisseria meningitidisis an obligate human pathogen that colonizes the human nasopharyngeal mucosa and occasionally invades the bloodstream, causing severe invasive disease such as septicemia or meningitis [1]. Countries of the African meningitis belt have for over 100 years experienced large meningococcal meningitis epidemics [24]. Multilocus sequence typing (MLST) of carriage and disease isolates has shown that between the late 1980s and 2010, epidemic waves of colonization and disease were mainly associated with hypervirulent serogroup A clones with the sequence types (ST) 5, 7, and 2859 [58]. The ST-5 clone spread in the meningitis belt after an epidemic in Mecca in 1987. The ST-7 and the ST-2859 clone were found in Africa for the first time in 1995 and 2003, respectively [911]. Comparative whole genome sequencing analyzes showed that this ST-2859 lineage AMG-458 evaded herd immunity elicited by their ST-7 ancestors primarily through rapid homologous replacement of a set of genomic loci affecting non-capsular cell surface components [12]. The closely related A clones, namely, ST-5, 7 and 2859 followed each other in different geographical settings, each colonizing the local population for a few years before being replaced [1315]. Due to horizontal gene transfer and point mutations, clonal meningococcal populations are, even in the course of an epidemic, not absolutely identical. Analysis of ST-7 and ST-2859 serogroup A isolates isolated between 2001 and 2009 in the framework of longitudinal meningococcal colonization and disease surveys in Ghana and Burkina Faso identified thepilT-pilUlocus, which is usually implicated in the regulation of type IV pilus expression, and thecsaABCD-ctrABCD-ctrEFcapsule synthesis locus (cps) as point mutation hot spot regions [12]. The capsular genes are clustered within thecpschromosomal locus and divided into six regions, designated A-D, D and E. Genes in region A (csaABCD) encode for polysaccharide biosynthesis and polymerization enzymes [16]. Proteins encoded by genes in region B (ctrE and ctrF) and C (ctrABCD) are involved in translocation of the polysaccharide from the cytoplasm to the cell surface. Region D encoded proteins are required for lipopolysaccharide (LPS) synthesis. Genes in region D encode methyltransferases and region D genes which are duplicated and truncated (galE). The function of region E is unknown. The serogroup A polysaccharide consists of repeating models of (1-6)-linkedN-acetyl-mannosamine-1-phosphate synthesized by enzymes encoded by genes designatedmynA-DorcsaA-D[1618]. The repeating models are O-acetylated mainly at position C3 and sometimes at position C4 [17]. Meningococci have developed a range of genetic mechanisms to modify expression and structure of their capsule [19]. These include phase variation by slipped-strand miss-pairing, transcriptional regulation, post-transcriptional regulation via RNA thermosensors, insertion element (Is usually) associated on-off switching and capsule switching by horizontal gene transfer. Among the 100 clonally related serogroup A isolates analyzed by comparative whole genome sequencing, Lamelas et al. [12] identified 13 isolates with non-synonymous single nucleotide polymorphisms (SNPs) or stop codons in the capsule synthesis locus. In contrast, none of the 100 sequenced isolates carried a synonymous point mutation in this locus, speaking for selection of variants. Here we describe the phenotypic, structural and functional effects caused by the non-synonymous point mutations and stop codons in thecpslocus. == Materials and methods == == Neisseria meningitidis isolates == The serogroup AN. meningitidisisolates investigated in this study (Table 1) have been collected in the framework of longitudinal meningococcal colonization and disease studies in the Kassena-Nankana District (KND) and the neighboring Bolgatanga district (BA) hospital of Ghana and in the Nouna Health District (NHD) of Burkina Faso. Case isolates were obtained from the cerebrospinal fluid of meningitis patients and carriage isolates from throat swabs. For the longitudinal meningococcal AMG-458 colonization studies AMG-458 with a sample size of about 300 participants, residential compounds in the KND and the NHD were selected and throat swabs were taken twice annually from all consenting members of the selected compounds present at the time of the visit [15,20,21]. Isolation and characterization of bacterial isolates [15,20,21], and the comparative whole genome analysis of these isolates [12] have been described previously. Ethical clearance was obtained from the relevant institutional review boards and the Ministries of Health. Informed consent for use of the samples was obtained from all study participants. == Table 1. == Serogroup.