Evolution and pathogenesis of the Neisseria meningitidis urethritis-causing clade, NmUC Restricted; Files Only
Rodriguez, Emilio (Fall 2024)
Abstract
Neisseria meningitidis (Nm), a pathogen exclusive to humans, is the causative agent of invasive meningococcemia and epidemic meningitis. While normally colonizing the human nasopharynx, a novel Nm clade has emerged in the last decade that is an effective male urethral pathogen resembling Neisseria gonorrhoeae (Ng) and designated NmUC. A previous analysis of over 200 NmUC isolates revealed the integration of Ng DNA into several genomic sites, including the Ng denitrification apparatus aniA–norB, a partial Ng operon NEIS1446-NEIS1442 (ispD), and Ng argB encoding the acetylglutamate kinase. Present in all NmUC isolates was an IS1301-mediated deletion of group C capsule genes, resulting in loss of surface capsule. We investigated the role of the NEIS1446-NEIS1442 (ispD) transfer in NmUC and found that ispD was essential in Nm and enhanced anaerobic survival of Nm. IspD, also essential in other gram-negative bacteria, catalyzes the third step in isoprenoid synthesis pathway, required for generation of ubiquinone that serves as the electron carrier in the electron transport chain. Using translational reporters, we showed that the integration of the gonococcal NEIS1446-NEIS1442 into the clade genome increased expression of ispD, with higher activities than non-clade Nm but comparable to Ng under both aerobic and microaerobic conditions in an NmUC background. The increased expression of ispD enhanced anaerobic survival, potentially by increasing production of ubiquinone and promoting the oxygen-independent electron transfer in oxygen-limited environments found in the urogenital tract. Further, we investigated the continued genomic evolution of NmUC using the genomes of newly identified clade isolates recovered globally since the original (2016) report. We found that over 90% of isolates maintained the key genomic features of NmUC, indicating the importance of these features to the clade’s biology. Phylogenetic analysis of all NmUC isolates revealed the emergence of a sub-clade (NmUC-B) that has acquired additional genomic features distinct from earlier clade isolates (NmUC-A), including fluoroquinolone resistance alleles from Ng. Fluoroquinolone resistance has been increasing in the broader Nm community and is affecting chemoprophylaxis options. This work furthers our understanding of the role of horizontal gene transfer from Ng to Nm contributing to NmUC emergence and evolution as a urogenital pathogen.
Table of Contents
Abstract
Acknowledgements
Table of Contents
Chapter 1: Introduction....................................................................................................... 1
Chapter 2: Contribution of the gonococcal NEIS1446-ispD gene conversion to the pathobiology of the Neisseria meningitidis urethritis clade, NmUC................................................................................................................................ 16
Figure 1: Gene conversion in the NEIS1446-NEIS1438 operon in NmUC........... 44
Figure 2: Generation of ispD deletion mutants and impact on aerobic growth.... 46
Figure 3: Amino acid sequence comparison between NmUC, Ng, and non-clade Nm IspD proteins 48
Figure 4: Aerobic growth curves of NmUC, Nm, Ng, and four ispD-complemented strains in GC broth 50
Figure 5: Survival of NmUC, Nm, and ispD-complemented strains under anaerobic conditions 52
Figure 6: Expression of NEIS1446-ispD............................................................... 54
Table S1: Strains used in this study....................................................................... 55
Table S2: Primers used in this study..................................................................... 56
Figure S1: Confirmation of the NEIS1446 (HP)-NEIS1438 (abcT) operon in NmUC 57
Figure S2: The crystal structure of E. coli IspD dimer complexed with CDP-ME and Mg2+ 59
Figure S3: IspD site-directed mutations do not affect the minimum inducer concentration required for robust growth 60
Figure S4: The complemented mutant with deleted native ispD has a low level of ispD transcription when uninduced, indicating a leaky promoter control.................................................................................................... 61
Chapter 3: Continuing genomic evolution of the Neisseria meningitidis cc11.2 urethritis clade, NmUC: a narrative review 63
Table 1: Frequencies of alleles in the initial 209 (2013-2016) NmUC-A isolates.....
............................................................................................................................... 91
Table 2: Allele frequency of characteristic NmUC genes in the 52 recent (2016-2022) clade isolates 93
Table 3: Identity and frequency of characteristic new SNP regions in the 40 NmUC-B sub-clade isolates 94
Table 4: Identity and frequency of alleles conferring increased antibiotic resistance in the 261 NmUC Isolates 97
Figure 1: The gonococcal denitrification apparatus in NmUC.............................. 98
Figure 2: SNP-based phylogeny of NmUC isolates............................................ 100
Figure 3: SNP density plots of new SNPs regions defining the 40 NmUC-B sub-clade isolates 101
Chapter 4: Progression of Antibiotic Resistance in Neisseria meningitidis.................... 102
Table 1: Genetic Changes Conferring Meningococcal Antibiotic Resistance.... 146
Table 2: Emergence of Penicillin Intermediate and Resistant N. meningitidis... 147
Table 3: Antibiotic Treatment of Invasive Meningococcal Disease in Adults and Children 149
Table 4a: Close Contacts of the Index Patients and Recommendations for Chemoprophylaxis 151
Table 4b: Antibiotic Chemoprophylaxis for Close Contacts defined in Table 4a.....
............................................................................................................................. 152
Figure 1: Phylogenetic distribution of high-level β-lactam and quinolone resistance in N. meningitidis 154
Figure S1: Global distribution of high-level penicillin-resistant Nm isolates..... 158
Figure S2: Global distribution of fluoroquinolone-resistant Nm isolates........... 160
Figure S3: Distribution of the single nucleotide polymorphisms (SNPs) in gyrA and parC conferring quinolone resistance in Nm 162
Chapter 5: Discussion...................................................................................................... 163
Figure 1: Model for the importance of the gonococcal NEIS1446-ispD gene conversion event in anaerobic survival of NmUC 177
Figure 2: Genomic features identical in historic and recent NmUC isolates...... 178
Figure 3: Antibiotic resistance alleles in NmUC................................................. 179
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