The O-acetylation of peptidoglycan (PG) is currently known to occur in greater than 50 eubacterial species, including numerous pathogens. This modification, which occurs at the C-6 hydroxyl of the N-acetylmuramoyl residues within the heteropolymer’s glycan backbone, serves as a cell wall autolytic regulatory mechanism, and contributes to pathogenesis and persistence within a host. Despite these significant physiological and pathobiological roles however, the identity of the pathway(s) responsible for the modification was only recently elucidated, for which two unrelated systems were identified, viz., the O-acetylpeptidoglycan (OAP) cluster-encoded multi-component system typical of Gram-negative species and the singular OatA of Gram-positives. As part of the OAP PG O-acetylation system, our group previously identified O-acetylpeptidoglycan esterase (Ape) as an enzyme responsible for the removal of the modification, permitting the continued metabolism of the PG sacculus. Herein, studies were performed to assess the postulated viability of this class of enzyme as a novel antibacterial target. Specifically, recombinant Ape1 from Neisseria gonorrhoeae was purified to homogeneity and the inhibitory effect of purpurin, a natural product identified as such, evaluated in detail. Kinetic analysis demonstrated that the compound elicited a competitive mode of inhibition (Kic ~3.7 μM), while the in vivo treatment of an array of environmental and pathogenic species was found to result in growth arrest for those cells containing both O-acetylPG and Ape. Evaluation of modification levels, cell wall morphology, and viability indicated a bacteriostatic effect. Taken together these data provide proof of principle that this class of enzyme presents a worthy therapeutic target. In addition to the presence of an Ape, the OAP system further differs from that of OatA through the use of two PG O-acetyltransferases. While purported to be mutually exclusive and evolutionarily divergent, in silico genomic analyses indicated their potential copresence in Bacillus anthracis and other closely related organisms. Indeed, purpurin-mediated differential growth inhibition between several such isolates and other bacilli indicated Ape activity therein. To investigate this possibility, the hypothetical Ape3 protein from Bacillus cereus ATCC 10987 was overproduced, purified, and its function assessed. Data from activity assays involving natural and synthetic substrates indicated that the protein possesses basal esterase activity in vitro. Phenotypic analysis of B. anthracis mutants deficient in each of the organism’s putative integral membrane PG O-acetyltranslocases subsequently indicated that Ape3 preferentially functions as a PG O-acetyltransferase (Pat) in vivo and that the OAP-mediated system is required for the separation of daughter cells following division. In addition, the presence of an Oat homologue was also confirmed. Thus, this is the first report of a bacterium known to possess both types of PG O-acetylation systems. / Natural Sciences and Engineering Research Council of Canada (NSERC)

Gonorrhea is caused by the human pathogen Neisseria gonorrhoeae. More than 106 million new cases of N. gonorrhoeae infections occur each year worldwide. There is no vaccine available against gonococcal infections and treatment of gonorrhea with antibiotics is the only way to eradicate infection. The high prevalence of antibiotic resistance (AMR) in this microorganism makes the effective treatment of gonococcal infections increasingly problematic. The emergence of AMR, especially to extended spectrum cephalosporins (i.e. cefixime and ceftriaxone) which are the last possibilities for single dose treatment options for gonococcal infections, is a serious concern. Gonorrhea may become an untreatable infection in the near future. Saskatchewan (SK) has one of the highest rates of gonorrhea in Canada. In order to better characterize the gonorrhea epidemic in SK, the objectives of the present research were to determine the prevalence and trends of AMR and emerging AMR mechanisms in N. gonorrhoeae isolates. AMR mechanisms were ascertained for the first time in SK in order to identify genetic causes of resistance. This was completed by determining and analyzing the DNA sequences of various genes - penA, mtrR, porB ponA, gyrA, parC mtrR, 23S rRNA alleles and erm –implicated in gonococcal AMR. The population dynamics of the N. gonorrhoeae isolates in SK was investigated by DNA based molecular methods to determine strain distribution, evolution of AMR phenotypes, and association between strain types (STs) and AMR genotypes and phenotypes. N. gonorrhoeae isolates (n=427) from Saskatchewan (2003-2011) were susceptible to antibiotics now recommended for treatment - cefixime, ceftriaxone and spectinomycin. Over 95% of the isolates tested were also susceptible to penicillin (96%) and ciprofloxacin (95.5%), antibiotics no longer recommended for treatment, and azithromycin (99.4%). Tetracycline resistance was also high (50.1%). N. gonorrhoeae isolates that were resistant to the antibiotics tested and also those isolates with MICs ≥0.003 mg/L to cefixime and ceftriaxone were analyzed (n=146) to determine their resistance mechanisms. This analysis revealed that reduced susceptibility to ceftriaxone and cefixime and resistance to penicillin is mediated by specific mutations in penicillin binding protein 2 (PBP2), in the promoter and dimerization domains of MtrR and porin protein (PorB). Novel mutations and combinations of mutations were noted. Ciprofloxacin resistant N. gonorrhoeae isolates carried double mutations in GyrA (S91F and D95G/N) and a S87R or S88P substitution in ParC. Isolates resistant to azithromycin had specific mutations in all the four alleles of 23S rRNA as well as in the DNA binding domain of MtrR. Most resistance was chromosomally mediated while plasmid-mediated resistance to penicillin (0.93% of penicillin resistant isolates) and tetracycline (3.3%) was low. DNA based strain typing methods such as porB-DNA sequencing, N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) showed that the gonococcal population in SK differs appreciably from both other Canadian provinces and from strains reported internationally. MLST analysis, which ascertains the evolution of isolates over time, demonstrated that penicillin and tetracycline resistant isolates in SK evolved through spontaneous mutations in established lineages. Ciprofloxacin and azithromycin resistant N. gonorrhoeae isolates, on the other hand, were introduced into SK from outside the province. Significant associations between particular mutation pattern combinations in resistance determining genes and specific NG-MAST STs were identified e.g. NG-MAST ST 25 was associated with specific combined mutation patterns in PBP2, MtrR and PorB and antibiotic susceptibility; and, NG-MAST ST 3654 was associated with another PBP2/MtrR/PorB mutation pattern, chromosomal resistance to penicillin and tetracycline and elevated MICs to cefixime. This research shows the importance of regional antimicrobial susceptibility monitoring. In the context of SK, this means that local surveillance of gonococcal AMR may be used to develop policies for regional treatment guidelines which promote the prudent use of antimicrobials for treatment, including those antibiotics which may no longer be used in other regions due to higher AMR rates. Further, the significant association between particular AMR mutation pattern combinations and specific STs indicates that AMR might be predicted. These results should assist in the development of non-culture-based tests for the diagnosis of gonococcal AMR similar to nucleic acid amplification tests used to diagnose N. gonorrhoeae infections.

The Neisseria genus includes pathogens, Neisseria gonorrhoeae (Ngo) and Neisseria meningitidis, as well as commensals. Ngo, the cause of gonorrhea, induces massive inflammation but a surprising lack of adaptive immune responses. We have observed that Ngo can inhibit both T cell activation and dendritic cell maturation through interaction with the host expressed co-inhibitory receptor carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1). Therefore, I wondered whether B cells may also be affected in this manner. Herein, I examine primary human B cell responses to infection with Ngo, as well as the other Neisseria species. B cells infected with Ngo show no sign of inhibition, regardless of their ability to bind CEACAM1, instead responding to gonococci with robust activation and proliferation. There are distinct subsets of B cells found in the periphery and, intriguingly, the IgM memory B cell subset expand and produce polyreactive IgM in response to goncoccal infection. These cells are innate in function, producing low affinity, polyclonal IgM that is protective against bacterial and fungal dissemination. This effect was broadly specific for Neisseria sp., as B cell infection with all commensal Neisseria species examined induced innate B cell responses. Curiously, meningococcal strains avoid inducing the innate B cell responses, making it enticing to hypothesize that its avoidance of such an ancient immune response may contribute to its ability to cause disease in humans. Finally, I tested whether gonococcal Opa protein binding to CEACAM1 affects primary human B cell activation, and show that no inhibition was observed. This absence of co-inhibitory function of neisserial-bound CEACAM1 may reflect inherent differences between distinctive cell types. Combined, the results in this thesis contribute new insight regarding the poorly characterized human IgM memory B cells, as well as to the function of CEACAM1 in lymphocytes.

Background: Clinical breakpoints and epidemiological cut-off values for N. gonorrhoeae azithromycin antimicrobial susceptibility testing have not been established. This study utilized existing minimum inhibitory concentration (MIC) data from CDC’s Gonococcal Isolate Surveillance Project (GISP) to establish epidemiological cut-off values for azithromycin and N. gonorrhoeae as determined by agar dilution. Methods: MIC distributions for the pooled dataset and each data year (2005-2012) were constructed. Epidemiological cut-off values were calculated using two methods. Method 1 considers the wild-type MIC distribution, the modal MIC for the distribution, and the inherent variability of the test (±1 twofold-dilution). Method 2 defines the epidemiological cut-off value as two twofold-dilutions higher than the MIC50. Results: Taking into consideration the wild-type MIC distributions and the inherent variability of the test, the epidemiological cut-off value chosen for the pooled dataset and each data year using Method 1 was ≤1.0 µg/mL. The MIC50 for the pooled dataset and each data year was 0.25 µg/mL. Two twofold-dilutions higher than the MIC50 (0.25 µg/mL) for the pooled dataset and each data year was 1.0 µg/mL. Discussion: The epidemiological cut-off values chosen using Methods 1 and 2 (≤1.0 µg/mL) were identical for the pooled dataset and each data year, indicating the epidemiological cut-off value has not changed from 2005-2012. The epidemiological cut-off value for N. gonorrhoeae azithromycin agar dilution antimicrobial susceptibility testing established during this study can be used to help set clinical breakpoints and identify isolates with reduced susceptibility to azithromycin.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

This thesis examined molecular changes in the quinolone-resistance determining regions (QRDRs) of the topoisomerase genes, gyrA and parC of Neisseria gonorrhoeae and Neisseria meningitidis and their contribution to fluoroquinolone resistance (FQR). Initially models of FQR emergence were developed from analysis of resistant mutants generated in vitro. The effects of the nature and order of sequential changes in GyrA and ParC on FQR were explored by correlating QRDR changes with ciprofloxacin minimum inhibitory concentration (MIC) determinations. The in vitro models were validated by comparisons of QRDR changes and MICs in two populations of wild-type FQR N. gonorrhoeae over a wide MIC range (0.09 to 24??g/mL), and in a wild type FQR meningococcus. The in vitro activities of three newer quinolones with differential activity on GyrA and ParC were compared with that of ciprofloxacin. Key findings were that the initial QRDR changes always occurred in gyrA and were the predominant influence on phenotypic expression of FQR. QRDR alterations were acquired sequentially and two GyrA and two ParC changes represented the full complement of changes observed in gonococci and two GyrA and one ParC change those in meningococci. GyrA alterations at Ser-91 in gonococci and Thr???91 in meningococci were pivotal for the development of further resistance. ParC changes required the presence of two GyrA alterations for any major impact on FQR. ParC substitutions, Ser-87???Arg and Glu-91???Gly in gonococci and Cys- 85???Asp and Glu-91???Lys in meningococci led to the expression of the highest FQR levels. Examination of FQR in wild-type meningococci was necessarily restricted, but analyses using the broader MIC range available in in-vitro-derived FQR meningococci (0.09 to 16??g/mL) revealed the first ParC changes in N. meningitidis. The study also redefined QRDR boundaries and described novel mutations within them. The nature of sequence changes in GyrA and ParC in FQR Neisseria also affected the relative activities of the three newer quinolones. Trovafloxacin was the most active quinolone in vitro but MIC differences with ciprofloxacin were mutation-dependent. Grepafloxacin and moxifloxacin were only slightly more active than ciprofloxacin in the presence of multiple QRDR changes. This thesis provides a comprehensive analysis of the relationship between QRDR alterations and FQR in N. gonorrhoeae and offers insights into the potential for FQR development in N. meningitidis.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 5 uppsatser.

Thesis (M.S.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)

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