PacBio Amplicon Sequencing Method To Measure Pilin Antigenic Variation Frequencies of Neisseria gonorrhoeae

ABSTRACT Gene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the nonexpressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences (PacBio) amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection. IMPORTANCE Diversity generation systems are used by many unicellular organism to provide subpopulations of cell with different properties that are available when needed. We have developed a method using the PacBio DNA sequencing technology and a custom computer program to analyze the pilin antigenic variation system of the organism that is the sole cause of the sexually transmitted infection, gonorrhea.

PacBio amplicon sequencing method to measure pilin antigenic variation frequencies of Neisseria gonorrhoeae

ABSTRACTGene diversification is a common mechanism pathogens use to alter surface structures to aid in immune avoidance. Neisseria gonorrhoeae uses a gene conversion-based diversification system to alter the primary sequence of the gene encoding the major subunit of the pilus, pilE. Antigenic variation occurs when one of the non-expressed 19 silent copies donates part of its DNA sequence to pilE. We have developed a method using Pacific Biosciences amplicon sequencing and custom software to determine pilin antigenic variation frequencies. The program analyzes 37 variable regions across the strain FA1090 1-81-S2 pilE gene and can be modified to determine sequence variation from other starting pilE sequences or other diversity generation systems. Using this method, we measured pilin antigenic variation frequencies for various derivatives of strain FA1090 and showed we can also analyze pilin antigenic variation frequencies during macrophage infection.

Pharmacokinetic Data Are Predictive ofIn VivoEfficacy for Cefixime and Ceftriaxone against Susceptible and ResistantNeisseria gonorrhoeaeStrains in the Gonorrhea Mouse Model

ABSTRACTThere is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESCs) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fTMIC]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESCr) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, within vivoefficacy against an ESCsstrain requiring doses that yielded anfTMICin excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESCrstrain and were useful in designing effective dosing regimens.

Assembly of NADPH Oxidase in Human Neutrophils Is Modulated by the Opacity-Associated Protein Expression State of Neisseria gonorrhoeae

ABSTRACTNeisseria gonorrhoeae(the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91phox/p22phox) and the cytoplasm (p47phox, p67phox, and p40phox). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa−) Gc does not stimulate neutrophil ROS production. Using constitutively Opa−and OpaD-positive (OpaD+) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa−Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD+Gc. gp91phoxand p22phoxwere recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa−Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47phox“organizing” subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.

Structure-Function Studies of the Neisseria gonorrhoeae Major Outer Membrane Porin

ABSTRACTThe major outer membrane porin (PorB) expressed byNeisseria gonorrhoeaeplays multiple roles during infection, in addition to its function as an outer membrane pore. We have generated a panel of mutants ofN. gonorrhoeaestrain FA1090 expressing a variety of mutantporBgenes that all function as porins. We identified multiple regions of porin that are involved in its binding to the complement regulatory factors C4b-binding protein and factor H and confirmed that the ability to bind at least one factor is required for FA1090 to survive the bactericidal effects of human serum. We tested the ability of these mutants to inhibit both apoptosis and the oxidative burst in polymorphonuclear leukocytes but were unable to identify the porin domains required for either function. This study has identified nonessential porin domains and some potentially essential portions of the protein and has further expanded our understanding of the contribution of the porin domains required for complement regulation.

Frequency and Rate of Pilin Antigenic Variation of Neisseria meningitidis

ABSTRACT The rates of pilin antigenic variation (Av) of two strains of Neisseria meningitidis were determined using an unbiased DNA sequencing assay. Strain MC58 underwent pilin Av at a rate similar to that of N. gonorrhoeae strain MS11 but lower than that of N. gonorrhoeae strain FA1090. Pilin Av was undetectable in strain FAM18.

Opacity Proteins Increase Neisseria gonorrhoeae Fitness in the Female Genital Tract Due to a Factor under Ovarian Control

ABSTRACT The neisserial opacity (Opa) proteins are a family of antigenically distinct outer membrane proteins that undergo phase-variable expression. Opa+ variants of Neisseria gonorrhoeae strain FA1090 are selected in a cyclical pattern from the lower genital tract of estradiol-treated mice. Here we show that cyclical recovery of Opa+ gonococci does not occur in ovariectomized mice; therefore, the reproductive cycle plays a role in the selection kinetics in vivo. As predicted by the selection pattern shown by wild-type gonococci, we demonstrated that a constitutive Opa-expressing strain was more fit than an Opa-deficient mutant in the early and late phases of infection. We found no evidence that Opa-mediated colonization selects for Opa+ variants during murine infection based on adherence assays with cultured murine epithelial cells. We also tested the hypothesis that complement selects for Opa protein expression during infection. Although some Opa+ variants of a serum-sensitive derivative of strain FA1090 were more resistant to the bactericidal activity of normal human serum, selection for Opa expression was not abrogated in C3-depleted mice. Finally, as previously reported, Opa+ gonococci were more sensitive to serine proteases. Thus, proteases or protease inhibitors may contribute to the observed in vivo selection pattern. We concluded that Opa proteins promote persistence of N. gonorrhoeae in the female genital tract and that opa gene phase variation allows gonococci to evade or capitalize upon unidentified host factors of the mammalian reproductive cycle. This work revealed an intimate interaction between pathogen and host and provides evidence that hormonally related factors shape bacterial adaptation.

Ecf, an Alternative Sigma Factor from Neisseria gonorrhoeae, Controls Expression of msrAB, Which Encodes Methionine Sulfoxide Reductase

ABSTRACT A DNA microarray was used to identify genes transcribed in Neisseria gonorrhoeae using Ecf, an alternative sigma factor. No differences between the transcriptional profiles of strain FA1090 and a mutant where ecf had been inactivated could be detected when both were grown in vitro. We therefore constructed a gonococcal strain in which Ecf can be overexpressed. Some differentially expressed genes are clustered with ecf on the genome and appear to form a single transcriptional unit. Expression of the gene encoding MsrAB, which possesses methionine sulfoxide reductase activity, was also dependent on Ecf, suggesting that the regulon responds to oxidative damage. Western blotting confirmed that the increased level of MsrAB protein is dependent on the presence of Ecf.

DNA methyltransferases from Neisseria meningitidis and Neisseria gonorrhoeae FA1090 associated with mismatch nicking endonucleases

The genes encoding the DNA methyltransferases M.NmeDI and M.NmeAI from Neisseria meningitidis associated with the genes encoding putative Vsr endonucleases were overexpressed in Escherichia coli. The enzymes were purified to apparent homogeneity on Ni-NTA agarose columns, yielding proteins of 49±1 kDa and 39·6±1 kDa, respectively, under denaturing conditions. M.NmeDI recognizes the degenerate sequence 5′-RCCGGB-3′. It methylates the first 5′ cytosine residue on both strands within the core sequence CCGG. The enzyme shows higher affinity with the hemimethylated degenerate sequence than with the unmethylated degenerate sequence. Comparison of the amino acid sequence of the target-recognizing domain of M.NmeDI with the closest neighbours recognizing the sequence 5′-RCCGGY-3′ showed the presence of the homologous domain and an additional domain that may be responsible for recognizing the degenerate sequence. M.NmeAI recognizes the sequence 5′-CCGG-3′ and methylates the second 5′ cytosine residue on both DNA strands. In Neisseria gonorrhoeae strain FA1090 the homologues of these ORFs are truncated due to a variety of mutations.

Genome Analysis and Strain Comparison of Correia Repeats and Correia Repeat-Enclosed Elements in Pathogenic Neisseria

ABSTRACT Whole genome sequences of Neisseria meningitidis strains Z2491 and MC58 and Neisseria gonorrhoeae FA1090 were analyzed for Correia repeats (CR) and CR-enclosed elements (CREE). A total of 533, 516, and 256 copies of CR and 270, 261, and 102 copies of CREE were found in these three genomes, respectively. The lengths of CREE range from 28 to 348 bp, and the lengths of multicopy CREE appear mainly in the ranges of 154 to 156 bp and 105 to 107 bp. The distribution of CREE lengths is similar between the two N. meningitidis genomes, with a greater number of 154- to 156-bp CREE (163 and 152 copies in N. meningitidis strain Z2491 and N. meningitidis strain MC58, respectively) than 105- to 107-bp CREE (72 and 77 copies). In the N. gonorrhoeae strain FA1090 genome there are relatively more 105- to 107-bp CREE (51 copies) than 154- to 156-bp CREE (36 copies). The genomic distribution of 107-bp CREE also shows similarity between the two N. meningitidis strains (15 copies share the same loci) and differences between N. meningitidis strains and N. gonorrhoeae FA1090 (only one copy is located in the same locus). Detailed sequence analysis showed that both the terminal inverted repeats and the core regions of CREE are composed of distinct basic sequence blocks. Direct TA dinucleotide repeats exist at the termini of all CREE. A survey of DNA sequence upstream of the sialyltransferase gene, lst, in several Neisseria isolates showed that 5 N. meningitidis strains contain a 107-bp CREE in this region but 25 N. gonorrhoeae strains show an exact absence of a 105-bp sequence block (i.e., the 107-bp CREE without a 5′ TA dinucleotide) in the same region. Whole-genome sequence analysis confirmed that this 105-bp indel exists in many homologous 107-bp CREE loci. Thus, we postulate that all CREE are made of target TA with indels of various lengths. Analysis of 107-bp CREE revealed that they exist predominantly in intergenic regions and are often near virulence, metabolic, and transporter genes. The abundance of CREE in Neisseria genomes suggests that they may have played a role in genome organization, function, and evolution. Their differential distribution in different pathogenic Neisseria strains may contribute to the distinct behaviors of each Neisseria species.