Implementing PathElective as an organized means of supplementing pathology education in an osteopathic medical school-the New York Institute of Technology College of Osteopathic Medicine experience.

Pathology is not traditionally chosen by medical students applying to residency. In osteopathic medical schools, limited access to dedicated pathology faculty further complicates this issue. Because of a lack of pathology experiences, osteopathic medical students may not be as familiar with a pathology career. The purpose of this brief report is to describe the pilot experience of implementing a pre-existing web-based, free virtual platform for pathology education as alternative, supplemental exposure to pathology for osteopathic medical students at our institution. We began to offer the online pathology elective for Academic Year 2022-2023. Using the online free service of PathElective, this course provided a valuable exposure to pathology with multiple modules in anatomic, clinical, and digital/molecular pathology, before and after assessments, recorded videos by pathology experts, handouts, and reading assignments. During the first week, three introductory modules were required followed by weeks 2-4, in which the students would complete a total of 10 modules of their own choice. In total, 14 students participated in this virtual rotation from August 2022-May 2023. All chose cardiac pathology as the most popular module. Three of the 14 students matched into pathology residencies. This small cohort of 4th year medical students at our osteopathic medical school successfully completed a virtual elective rotation with the resources of PathElective. We report the success of this experience and hope to continue monitoring progress.

Development of an In Vitro Test Method to Replace an Animal-Based Potency Test for Pertactin Antigen in Multivalent Vaccines.

There is increasing interest to replace animal-based potency assays used routinely to test vaccines, since they are highly variable, are costly, and present ethical concerns. The development of relevant in vitro assays is part of the solution. Using pertactin (PRN) antigen as an example in DTaP-IPV (diphtheria, tetanus, acellular pertussis, and inactivated poliovirus) vaccines, a PRN antigenicity ELISA was developed using two monoclonal antibodies with a high affinity to unique PRN epitopes, relevance to human immune responses, and evidence of functionality. The ELISA measured consistent PRN antigenicity between the vaccine lots and was validated to demonstrate its accuracy, precision, linearity, and specificity. Notably, the PRN antigenicity ELISA was more sensitive than the mouse-based potency test and could more effectively differentiate between degraded and intact vaccine lots compared to the in vivo test. From these studies, the PRN antigenicity ELISA is proposed as an in vitro replacement for the in vivo potency test for PRN in DTaP-IPV-based formulations. Important considerations in this study included comprehensive antibody characterization, testing of multiple vaccine lots, method validation, and comparison to animal-based potency. Together, these factors form part of an overall strategy that ensures reliable and relevant in vitro assays are developed to replace animal tests.

Evaluating the immunogenicity of an intranasal vaccine against nicotine in mice using the Adjuvant Finlay Proteoliposome (AFPL1).

Tobacco smoking is recognized as a global pandemic resulting in 6 million deaths per year. Despite a variety of anti-smoking products available to aid with tobacco cessation, the majority of people who attempt to quit smoking relapse within 6 months due to the addictive nature of nicotine. An immunotherapy approach could offer a promising treatment option by inducing a potent selective antibody response against nicotine in order to block its distribution to the brain and its addictive effects in the central nervous system. Our nicotine vaccine candidate was administered intranasally using the Neisseria meningitidis serogroup B Adjuvant Finlay Proteoliposome 1 (AFPL1) as a part of the delivery system. This system was designed to generate a robust immune response by stimulating IL-1ß production through Toll-like receptor 4 (TLR4), a potent mechanism for mucosal immunity. The vaccine induced high antibody titers in mice sera in addition to inducing mucosal antibodies. The efficacy of our vaccine was demonstrated using in vivo challenge experiments with radioactive [(3)H]-nicotine, followed by an analysis of nicotine distribution in the lung, liver, blood and brain. Our results were encouraging as the nicotine concentration in the brain tissue of mice vaccinated with our candidate vaccine was four times lower than in non-vaccinated controls; suggesting that the anti-nicotine antibodies were able to block nicotine from crossing the blood brain barrier. In summary, we have developed a novel nicotine vaccine for the treatment of tobacco addiction by intranasal administration and also demonstrated that the AFPL1 can be used as a potential adjuvant for this vaccine design.

Antimicrobial susceptibility of Canadian isolates of Helicobacter pylori in Northeastern Ontario.

BACKGROUND: Helicobacter pylori plays a significant role in gastritis and ulcers. It is a carcinogen as defined by the WHO, and infection can result in adenocarcinomas and mucosa-associated lymphoid tissue lymphomas. In Canada, rates of antimicrobial resistance are relatively unknown, with very few studies conducted in the past 15 years. OBJECTIVE: To examine rates of resistance in Sudbury, Ontario, compare antimicrobial susceptibility methods and attempt to determine the molecular basis of antibiotic resistance. METHODS: Patients attending scheduled visits at Health Sciences North (Sudbury, Ontario) provided gastric biopsy samples on a volunteer basis. In total, 20 H pylori isolates were collected, and antimicrobial susceptibility testing (on amoxicillin, tetracycline, metronidazole, ciprofloxacin, levofloxacin and clarithromycin) was conducted using disk diffusion and E-test methods. Subsequently, genomic DNA from these isolates was sequenced to detect mutations associated with antimicrobial resistance. RESULTS: Sixty-five percent of the isolates were found to be resistant to at least one of the listed antibiotics according to E-test. Three isolates were found to be resistant to ≥3 of the above-mentioned antibiotics. Notably, 25% of the isolates were found to be resistant to both metronidazole and clarithromycin, two antibiotics that are normally prescribed as part of first-line regimens in the treatment of H pylori infections in Canada and most of the world. Among the resistant strains, the sequences of 23S ribosomal RNA and gyrA, which are linked to clarithromycin and ciprofloxacin/levofloxacin resistance, respectively, revealed the presence of known point mutations associated with antimicrobial resistance. CONCLUSIONS: In general, resistance to metronidazole, ciprofloxacin/levofloxacin and clarithromycin has increased since the studies in the early 2000s. These results suggest that surveillance programs of H pylori antibiotic resistance may need to be revisited or improved to prevent antimicrobial therapy failure.

HISTORIQUE: L'Helicobacter pylori contribue énormément à la gastrite et aux ulcères. L'OMS le définit comme un cancérigène, et l'infection peut provoquer l'apparition d'adénocarcinomes et de lymphomes de tissus lymphoïdes associés aux muqueuses. Au Canada, on connaît relativement peu les taux de résistance antimicrobienne, car très peu d'études ont été réalisées sur le sujet depuis 15 ans. OBJECTIF: Examiner les taux de résistance à Sudbury, en Ontario, comparer les méthodes de susceptibilité antimicrobienne et tenter de déterminer le fondement biologique de la résistance antibiotique. MÉTHODOLOGIE: Les patients qui allaient à un rendez-vous prévu au Health Sciences North de Sudbury ont remis les résultats de biopsies gastriques sur une base volontaire. Au total, 20 isolats de H pylori ont été recueillis, et les tests de susceptibilité antimicrobienne (à l'amoxicilline, à la tétracycline, au métronidazole, à la ciprofloxacine, à la lévofloxacine et à la clarithromycine) ont été effectués au moyen de la diffusion sur disque et de l'essai E. L'ADN génomique de ces isolats a ensuite été séquencé pour déceler les mutations associées à la résistance antimicrobienne. RÉSULTATS: Selon l'essai E, 65 % des isolats étaient résistants à au moins un des antibiotiques énumérés. Notamment, 25 % des isolats étaient résistants à la fois au métronidazole et à la clarithromycine, tous deux normalement prescrits en première ligne pour traiter les infections à H pylori au Canada et dans la plupart des régions du monde. Parmi les souches résistantes, les séquences d'ARN ribosomique 23S et de gyrA, qui sont liées à la résistance à la clarithromycine et à la ciprofloxacine-lévofloxacine, respectivement, révélaient la présence de mutations de points connus associés à une résistance antimicrobienne. CONCLUSIONS: En général, la résistance au métronidazole, à la ciprofloxacine-lévofloxacine et à la clarithromycine a augmenté depuis les études réalisées au début des années 2000. D'après ces résultats, il faudra peut-être revoir ou améliorer les programmes de surveillance de l'antibiorésistance au H pylori pour prévenir l'échec du traitement antimicrobien.

Caspase-1 Dependent IL-1ß Secretion and Antigen-Specific T-Cell Activation by the Novel Adjuvant, PCEP.

The potent adjuvant activity of the novel adjuvant, poly[di(sodiumcarboxylatoethylphenoxy)phosphazene] (PCEP), with various antigens has been reported previously. However, very little is known about its mechanisms of action. We have recently reported that intramuscular injection of PCEP induces NLRP3, an inflammasome receptor gene, and inflammatory cytokines, including IL-1ß and IL-18, in mouse muscle tissue. Caspase-1 is required for the processing of pro-forms of IL-1ß and IL-18 into mature forms and is a critical constituent of the NLRP3 inflammasome. Hence, in the present study, we investigated the role of caspase-1 in the secretion of IL-1ß and IL-18 in PCEP-stimulated splenic dendritic cells (DCs). Caspase inhibitor YVAD-fmk-treated splenic DCs showed significantly reduced IL-1ß and IL-18 secretion in response to PCEP stimulation. Further, PCEP had no effect on the expression of MHC class II or co-stimulatory molecules, CD86 and CD40, suggesting that PCEP does not induce DC maturation. However, PCEP directly activated B-cells to induce significant production of IgM. In addition, PCEP+ovalbumin (OVA) immunized mice showed significantly increased production of antigen-specific IFN-γ by CD4⁺ and CD8⁺ T-cells. We conclude that PCEP activates innate immunity, leading to increased antigen-specific T-cell responses.

The potential of 1018 ISS adjuvant in hepatitis B vaccines: HEPLISAV™ review.

Hepatitis B (HBV) virus infects the liver, and upon chronic infection, can cause liver cirrhosis and hepatocellular carcinoma. Despite universal vaccination programs against the virus, HBV still affects over 2 billion people worldwide, with over 240 million developing a chronic infection. While current alum-adjuvanted vaccines have shown efficacy in promoting seroprotection in healthy adults, 5-10% of immune-competent populations fail to achieve long-lasting seroprotection from these formulations. Furthermore, a large proportion of immunocompromised patients fail to achieve seroprotective antibody titers after receiving these vaccines. A novel vaccine candidate, HEPLISAV™, uses immunostimulatory sequences (ISS), in its formulation that helps induce a robust humoral and cell mediated immunity against HBV. In Phase III clinical trials, HEPLISAV™ has been shown to elicit seroprotective antibody titers with fewer immunizations. Similar safety profiles are demonstrated when compared with current HBV vaccines. For these reasons, HEPLISAV™ is an attractive vaccine to combat this global disease.

The recombinant globular head domain of the measles virus hemagglutinin protein as a subunit vaccine against measles.

Despite the availability of live attenuated measles virus (MV) vaccines, a large number of measles-associated deaths occur among infants in developing countries. The development of a measles subunit vaccine may circumvent the limitations associated with the current live attenuated vaccines and eventually contribute to global measles eradication. Therefore, the goal of this study was to test the feasibility of producing the recombinant globular head domain of the MV hemagglutinin (H) protein by stably transfected human cells and to examine the ability of this recombinant protein to elicit MV-specific immune responses. The recombinant protein was purified from the culture supernatant of stably transfected HEK293T cells secreting a tagged version of the protein. Two subcutaneous immunizations with the purified recombinant protein alone resulted in the production of MV-specific serum IgG and neutralizing antibodies in mice. Formulation of the protein with adjuvants (polyphosphazene or alum) further enhanced the humoral immune response and in addition resulted in the induction of cell-mediated immunity as measured by the production of MV H-specific interferon gamma (IFN-γ) and interleukin 5 (IL-5) by in vitro re-stimulated splenocytes. Furthermore, the inclusion of polyphosphazene into the vaccine formulation induced a mixed Th1/Th2-type immune response. In addition, the purified recombinant protein retained its immunogenicity even after storage at 37°C for 2 weeks.

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis.

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel(®) immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.

Combination adjuvants: the next generation of adjuvants?

Adjuvants are critical components of many vaccines. The majority of existing vaccines contain a single adjuvant. Owing to their inherent limitations, no single adjuvant is capable of inducing all the protective immune responses required in the many different vaccines. Consequently, investigators are exploring the potential of using formulations with multiple adjuvants in a vaccine. An emerging paradigm is that careful selection of adjuvant combinations can result in complementary and even synergistic enhancement of immune responses to vaccines. This approach is promising and presents tremendous opportunities for vaccinologists to tailor immune responses to specific vaccines. In this article, adjuvant combinations at different stages of development will be reviewed.

PCPP (poly[di(carboxylatophenoxy)-phosphazene]) microparticles co-encapsulating ovalbumin and CpG oligo-deoxynucleotides are potent enhancers of antigen specific Th1 immune responses in mice.

We generated poly[di(carboxylatophenoxy)-phosphazene] (PCPP) microparticles encapsulating ovalbumin (OVA) and CpG of 0.5-2.5 µm in diameter with an encapsulation efficiency of approximately 63% and 95% respectively. In mice the microparticles generated high antigen-specific IgG, IgG1 and IgG2a titers with higher IgG2a/IgG1 ratios. Whole body in vivo imaging of mice subcutaneously injected with MPs showed several fold increase of OVA and CpG in draining inguinal lymph nodes compared to soluble formulations. We conclude that PCPP MPs are more effective in enhancing immune responses compared to soluble formulations, due to co-delivery of OVA and CpG resulting in a Th1 type of immune response.

PCEP enhances IgA mucosal immune responses in mice following different immunization routes with influenza virus antigens.

BACKGROUND: We previously demonstrated that polyphosphazenes, particularly PCEP, enhance immune responses in mice immunized subcutaneously and intranasally. The objective of the present study was to investigate the efficacy of polyphosphazenes as adjuvants when delivered through different routes of vaccine administration. METHODS: BALB/c mice were immunized through intranasal, subcutaneous, oral and intrarectal delivery with vaccine formulations containing either influenza X:31 antigen alone or formulated in PCEP. Serum and mucosal washes were collected and assayed for antigen-specific antibody responses by ELISA, while splenocytes were assayed for antigen-specific cytokine production by ELISPOT. RESULTS: Intranasal immunization with PCEP+X:31 induced significantly higher IgA titers in all mucosal secretions (lung, nasal, and vaginal) compared to the other routes. Serum analysis showed that all mice given the PCEP+X:31 combination showed evidence of enhanced IgG2a titers in all administered routes, indicating that PCEP can be effective as an adjuvant in enhancing systemic immune responses when delivered via different routes of administration. CONCLUSIONS: We conclude that PCEP is a potent and versatile mucosal adjuvant that can be administered in a variety of routes and effectively enhances systemic and local immune responses. Furthermore, intranasal immunization was found to be the best administration route for enhancing IgA titers, providing further evidence for the potential of PCEP as a mucosal adjuvant.

The potential of polyphosphazenes for delivery of vaccine antigens and immunotherapeutic agents.

Polyphosphazene polyelectrolytes are synthetic, biodegradable polymers that have shown great potential in vaccine and drug delivery applications. Numerous investigations in laboratory animals have revealed that polyphosphazenes are also potent immunological adjuvants that can dramatically enhance the magnitude, quality and duration of immune responses to a variety of bacterial and viral vaccine antigens. Evidence is accumulating that these polymers have potent adjuvant activity in large animals as well. Interestingly, polyphosphazenes can be combined with novel immune modulatory agents resulting in even more potent immune activity and protection against experimental infection. While most reports are on the activity of polyphosphazenes in aqueous formulations, these polymers can also be easily made into microparticles, making them especially attractive for mucosal delivery. The mechanisms which mediate the adjuvant activity of polyphosphazenes are not fully understood, but there is evidence to suggest that activation of innate immunity may be involved. Further research and development of polyphosphazene adjuvants is warranted to fully explore their potential in the delivery of vaccines and immunotherapeutic agents.

Clusters of circulating Neisseria gonorrhoeae strains and association with antimicrobial resistance in Shanghai.

OBJECTIVES: (i) To distinguish Neisseria gonorrhoeae isolates in Shanghai by porB typing; (ii) to ascertain the congruence of porB DNA sequence typing with cases linked epidemiologically; (iii) to determine the association of specific PorB mutations with antimicrobial resistance to penicillin or tetracycline. METHODS: porB DNA sequences of 174 N. gonorrhoeae isolates, collected from 143 male patients and 31 female sexual partners in Shanghai were determined. Phylogenetic analysis was used to determine sequence associations and concordance with epidemiologically linked cases. PorB protein sequences were compared with the wild-type sequence to identify mutations associated with antimicrobial resistance to penicillin and tetracycline. RESULTS: porB1a genotypes comprised 27.0% of the isolates and included 15 distinct DNA sequences, while 73.0% of the isolates carried porB1b genotypes with 63 distinct DNA sequences. porB DNA sequence typing was congruent with patient-reported sexual contacts. In addition, porB DNA sequence analysis revealed a number of strains with identical DNA sequences not identified through traditional epidemiological methods. The porB1b isolates had a significantly higher percentage of chromosomally mediated resistance to tetracycline and higher MIC50s to penicillin and ciprofloxacin. G120K/A121D mutations were observed in 71.1% of PIB isolates and were associated with resistance to penicillin and/or tetracycline. The majority of the PIA isolates (82.1%) also carried G120D/A121G double mutations. The index of discrimination for porB DNA sequence analysis was 95%. CONCLUSIONS: The porB1b genotype was found to be predominant in Shanghai. porB DNA sequence typing was sufficiently discriminatory for differentiating N. gonorrhoeae isolates and was congruent with epidemiological linkages. Novel porB sequences of N. gonorrhoeae and novel mutations of PorB proteins were identified.

Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai.

OBJECTIVES: To determine the antimicrobial susceptibility of Neisseria gonorrhoeae from Shanghai and to type the quinolone resistance-determining regions (QRDRs) of ciprofloxacin-resistant isolates. METHODS: N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients in Shanghai and examined for their antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone. The mutation profiles of the QRDRs of gyrA and parC were determined for 103 isolates including one susceptible isolate and one isolate with intermediate levels of susceptibility to ciprofloxacin. RESULTS: High percentages of the 159 isolates were resistant to ciprofloxacin (98.7%), penicillin (93.1%) and tetracycline (56.5%). Penicillinase-producing N. gonorrhoeae (PPNG, 37.8%) or penicillinase-producing/tetracycline-resistant N. gonorrhoeae (PP/TRNG, 13.8%) accounted for 51.6% of the isolates. Chromosomal resistance to penicillin was observed in 41.5% of the isolates. Tetracycline resistance was noted in 56.5% of the isolates with 20.1% carrying plasmid-mediated resistance and 36.4% being chromosomally resistant. All isolates were susceptible to ceftriaxone and spectinomycin, although a trend to decreased susceptibility was noted. QRDR mutations were observed in the 101 ciprofloxacin-resistant isolates and the one ciprofloxacin-intermediate isolate, in contrast to the ciprofloxacin-susceptible isolate tested. Mutations in the QRDRs comprised four predominant (65.0% of the 103 isolates) patterns of a total of 19 patterns. Mutations in parC were significantly associated with higher MICs of ciprofloxacin. CONCLUSIONS: Spectinomycin and ceftriaxone are currently recommended for the treatment of gonorrhoea in Shanghai. Although the present study indicates that these antimicrobials should remain effective, the identification of isolates with decreased susceptibility underscores the importance of ongoing antimicrobial susceptibility surveillance to monitor and respond to the emergence of resistant isolates.

Conformation of the cell division regulator MinE: evidence for interactions between the topological specificity and anti-MinCD domains.

Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.

The C-terminus of MinE from Neisseria gonorrhoeae acts as a topological specificity factor by modulating MinD activity in bacterial cell division.

MinE regulates the proper placement of the cytokinetic FtsZ ring at midcell by inducing the pole-to-pole movement of MinCD complexes. While the N-terminus of MinE has been implicated in MinD binding, a clear functional role of the C-terminus has not been elucidated. We previously determined that MinE from Neisseria gonorrhoeae (Ng) was functional in Escherichia coli (Ec). Thus, using E. coli as a model organism, gonococcal MinE (MinE(Ng)) function was examined by generating amino acid substitutions of highly conserved MinE(Ng) residues and by testing the ability of the mutant proteins to interact with gonococcal MinD (MinD(Ng)), to induce a minicell phenotype upon overexpression, to initiate MinD(Ng) oscillation, and to stimulate MinD(Ng) ATPase activity. N-terminal MinE(Ng) mutants were unable to bind to MinD(Ng); thus, they did not induce a minicell phenotype, promote MinD(Ng) oscillation or stimulate MinD(Ng) ATPase activity. While C-terminal MinE(Ng) mutants exhibited reduced abilities to bind to MinD(Ng), we show that differences in MinD(Ng) binding to the C-terminus of MinE(Ng) alter the ability of MinE(Ng) to properly stimulate MinD(Ng) activity. We present four major findings from our studies of MinE(Ng): both the N- and C-termini of MinE(Ng) interact with MinD(Ng); interaction between MinD(Ng) and MinE(Ng) is required for the recruitment of MinD(Ng) to the coiled array; oscillation of MinD(Ng) does not require ATPase stimulation; and, the extent of MinD(Ng) ATPase stimulation depends on the binding strength between MinD(Ng) and the C-terminus of MinE(Ng.).

A conserved polar region in the cell division site determinant MinD is required for responding to MinE-induced oscillation but not for localization within coiled arrays.

A region in the cell division site determinant MinD required for stimulation by MinE and which determines MinD topological specificity along coil-like structures has been identified. Structural modeling of dimeric MinD and sequence alignment of 24 MinD proteins revealed a conserved polar region in Gram-negative bacterial MinD proteins, corresponding to residues 92-94 of Neisseria gonorrhoeae MinD (MinD(Ng)). Using MinD(Ng) as a paradigm for MinD functionality in Gram-negative organisms, mutation of these conserved residues did not abrogate MinD(Ng) self-association, nor its interaction with MinE(Ng) and the cell division inhibitor MinC. Although the MinD(Ng) mutant dimerized in the presence of ATP, its ATPase activity was not stimulated by MinE(Ng), unlike wild-type MinD(Ng). GFP fusions to either MinD(Ng) or to Escherichia coli MinD bearing simultaneous or individual mutations to residues 92-94 localized within coiled arrays along the E. coli inner cell periphery, similar to wild-type GFP-MinD. However, unlike wild-type GFP-fusions, the mutant proteins were distributed uniformly throughout the array, despite the presence of MinE, which normally imparts topological specificity to MinD by inducing the latter to oscillate from pole-to-pole and away from midcell. Hence, despite localizing along the inner cell periphery as a polymeric structure, the mutant MinD proteins in this study have lost the ability to be efficiently stimulated by MinE(Ng), resulting in a loss of distinct pole-to-pole oscillation.

The N terminus of MinD contains determinants which affect its dynamic localization and enzymatic activity.

MinD is involved in regulating the proper placement of the cytokinetic machinery in some bacteria, including Neisseria gonorrhoeae and Escherichia coli. Stimulation of the ATPase activity of MinD by MinE has been proposed to induce dynamic, pole-to-pole oscillations of MinD in E. coli. Here, we investigated the effects of deleting or mutating conserved residues within the N terminus of N. gonorrhoeae MinD (MinD(Ng)) on protein dynamism, localization, and interactions with MinD(Ng) and with MinE(Ng). Deletions or mutations were generated in the first five residues of MinD(Ng), and mutant proteins were evaluated by several functional assays. Truncation or mutation of N-terminal residues disrupted MinD(Ng) interactions with itself and with MinE. Although the majority of green fluorescent protein (GFP)-MinD(Ng) mutants could still oscillate from pole to pole in E. coli, the GFP-MinD(Ng) oscillation cycles were significantly faster and were accompanied by increased cytoplasmic localization. Interestingly, in vitro ATPase assays indicated that MinD(Ng) proteins lacking the first three residues or with an I5E substitution possessed higher MinE(Ng)-independent ATPase activities than the wild-type protein. These results indicate that determinants found within the extreme N terminus of MinD(Ng) are implicated in regulating the enzymatic activity and dynamic localization of the protein.