ParA: A Novel Target for Anti-Tubercular Drug Discovery

Tuberculosis (Tb) continues to be one of the world's greatest challenges in the public health arena. The current treatment for Tb entails a long duration of therapy making adherence to the whole course difficult. This has given rise to drug resistant strains of Mycobacterium tuberculosis which are posing a significant threat to Tb control strategies. To counteract this problem, there is an urgent need to develop alternative anti-tuberculous drugs which target processes that are critical for the growth and/or survival of this microbe. To identify such targets in M. tuberculosis, I used comparative genomics and mutagenesis data to identify conserved essential genes as viable targets for the development of broad-spectrum antibiotics. In addition, I validated the essentiality of three cell division genes in Mycobacterium smegmatis using conditional antisense RNA expression under different culture conditions. Furthermore, I performed high-throughput screens (HTS) using a differential susceptibility assay against one of the validated targets to identify its cognate inhibitor(s). Lastly, I developed a novel biochemical assay of the target to validate the specificity of the inhibitors identified in the HTS and evaluated the potency of the inhibitors against M. tuberculosis. This study identified 261 conserved putative essential genes as broad-spectrum targets. I hypothesized that antisense RNA expression of such genes will lead to its down-regulation and thereby affect the viability of the cells if these genes are essential. I also hypothesized that an essential gene will be required under all culture conditions. One gene, parA, demonstrated that it was essential under various culture conditions. This gene encodes for a protein which contain the conserved Walker A motif thus I theorized that it may posses ATPase activity. The results illustrated that the M. tuberculosis ParA protein possesses ATPase activity. This biochemical activity was used to validate two specific inhibitors of ParA, phenoxybenzamine and octoclothepin, which were identified in the cell-based HTS. Kinetic studies suggest that phenoxybenzamine is a mixed inhibitor while octoclothepin is a competitive inhibitor of ParA. This data is also supported by in silico docking. Both these compounds show low minimum inhibitory concentrations in M. smegmatis under nitrogen starvation conditions. In summary, this thesis illustrates that ParA is a viable target for anti-tubercular drugs. It demonstrates that ParA is an ATPase which has the potential to bind competitive and non-competitive inhibitors that can be exploited to target cell division in M. tuberculosis. Finally, this study presents phenoxybenzamine and octoclothepin as inhibitors of ParA. In conclusion, these compounds can either be developed to increase potency or be used as reference structures to screen for more potent inhibitors of the enzyme

Still 'dairy farm fever'? A Bayesian model for Leptospirosis notification data in New Zealand

Routinely collected public health surveillance data are often partially complete, yet remain a useful source by which to monitor incidence and track progress during disease intervention. In the 1970s, leptospirosis in New Zealand (NZ) was known as ‘dairy farm fever’ and the disease was frequently associated with serovars Hardjo and Pomona. To reduce infection, interventions such as vaccination of dairy cattle with these two serovars was implemented. These interventions have been associated with significant reduction in leptospirosis incidence, however, livestock-based occupations continue to predominate notifications. In recent years, diagnosis is increasingly made by nucleic acid detection which currently does not provide serovar information. Serovar information can assist in linking the recognized maintenance host, such as livestock and wildlife, to infecting serovars in human cases which can feed back into the design of intervention strategies. In this study, confirmed and probable leptospirosis notification data from 1 January 1999 to 31 December 2016 were used to build a model to impute the number of cases from different occupational groups based on serovar and month of occurrence. We imputed missing occupation and serovar data within a Bayesian framework assuming a Poisson process for the occurrence of notified cases. The dataset contained 1430 notified cases, of which 927 had a specific occupation (181 dairy farmers, 45 dry stock farmers, 454 meatworkers, 247 other) while the remaining 503 had non-specified occupations. Of the 1430 cases, 1036 had specified serovars (231 Ballum, 460 Hardjo, 249 Pomona, 96 Tarassovi) while the remaining 394 had an unknown serovar. Thus, 47% (674/1430) of observations had both a serovar and a specific occupation. The results show that although all occupations have some degree of under-reporting, dry stock farmers were most strongly affected and were inferred to contribute as many cases as dairy farmers to the burden of disease, despite dairy farmer being recorded much more frequently. Rather than discard records with some missingness, we have illustrated how mathematical modelling can be used to leverage information from these partially complete cases. Our finding provides important evidence for reassessing the current minimal use of animal vaccinations in dry stock. Improving the capture of specific farming type in case report forms is an important next step

Modifying Culture Conditions in Chemical Library Screening Identifies Alternative Inhibitors of Mycobacteria▿

In this study, application of a dual absorbance/fluorescence assay to a chemical library screen identified several previously unknown inhibitors of mycobacteria. In addition, growth conditions had a significant effect on the activity profile of the library. Some inhibitors such as Se-methylselenocysteine were detected only when screening was performed under nutrient-limited culture conditions as opposed to nutrient-rich culture conditions. We propose that multiple culture condition library screening is required for complete inhibitory profiling and for maximal antimycobacterial compound detection

“We don't really do doctors.” messages from people diagnosed with occupational leptospirosis for medical professionals on infection, hospitalisation, and long-term effects

Leptospirosis is largely an occupational disease for people working with livestock in Aotearoa New Zealand. Introduction of livestock vaccination and use of personal protective equipment has been associated with a reduction in the incidence. However, the incidence of occupational leptospirosis remains high, with significant burdens for affected families and healthcare system. For this article, a subset of thirteen participants from a nationwide leptospirosis case-control study (2019–2021) who were diagnosed with leptospirosis and worked with livestock at the time of illness were invited and agreed to a semi-structured interview. Interviewees reflected on their experiences as messages for medical professionals. The analysis of transcripts reveals widely shared experiences with infection, hospitalisation, and treatment, as well as long-term effects and recovery. Conclusions for medical professionals include that ill workers continue to have their diagnosis of leptospirosis delayed. This delay may contribute to more than half the people ill with leptospirosis hospitalised. Further, medical professionals' communication and relationship with ill people strongly colours the latter's experience, for good or for bad. Moreover, most interviewees experienced a recovery process that took several months of feeling tired, which undermined professional performance and emotional wellbeing

Leptospirosis in Aotearoa New Zealand: Protocol for a Nationwide Case-Control Study

International audienceBackground In Aotearoa New Zealand, 90% of patients with notified leptospirosis (a zoonotic bacterial disease) have been men working in agricultural industries. However, since 2008, the epidemiology of notified cases has been gradually changing, that is, more women are affected; there are more cases associated with occupations traditionally not considered high risk in New Zealand; infecting serovars have changed; and many patients experience symptoms long after infection. We hypothesized that there is a shift in leptospirosis transmission patterns with substantial burden on affected patients and their families. Objective In this paper, we aimed to describe the protocols used to conduct a nationwide case-control study to update leptospirosis risk factors and follow-up studies to assess the burden and sources of leptospirosis in New Zealand. Methods This study used a mixed methods approach, comprising a case-control study and 4 substudies that involved cases only. Cases were recruited nationwide, and controls were frequency matched by sex and rurality. All participants were administered a case-control questionnaire (study 1), with cases being interviewed again at least 6 months after the initial survey (study 2). A subset of cases from two high-risk populations, that is, farmers and abattoir workers, were further engaged in a semistructured interview (study 3). Some cases with regular animal exposure had their in-contact animals (livestock for blood and urine and wildlife for kidney) and environment (soil, mud, and water) sampled (study 4). Patients from selected health clinics suspected of leptospirosis also had blood and urine samples collected (study 5). In studies 4 and 5, blood samples were tested using the microscopic agglutination test to test for antibody titers against Leptospira serovars Hardjo type bovis, Ballum, Tarassovi, Pomona, and Copenhageni. Blood, urine, and environmental samples were also tested for pathogenic Leptospira DNA using polymerase chain reaction. Results Participants were recruited between July 22, 2019, and January 31, 2022, and data collection for the study has concluded. In total, 95 cases (July 25, 2019, to April 13, 2022) and 300 controls (October 19, 2019, to January 26, 2022) were interviewed for the case-control study; 91 cases participated in the follow-up interviews (July 9, 2020, to October 25, 2022); 13 cases participated in the semistructured interviews (January 26, 2021, to January 19, 2022); and 4 cases had their in-contact animals and environments sampled (October 28, 2020, and July 29, 2021). Data analysis for study 3 has concluded and 2 manuscripts have been drafted for review. Results of the other studies are being analyzed and the specific results of each study will be published as individual manuscripts.. Conclusions The methods used in this study may provide a basis for future epidemiological studies of infectious diseases. International Registered Report Identifier (IRRID) DERR1-10.2196/4790

The E. coli effector protein NleF is a caspase inhibitor.

Enterohemorrhagic and enteropathogenic E. coli (EHEC and EPEC) can cause severe and potentially life-threatening infections. Their pathogenicity is mediated by at least 40 effector proteins which they inject into their host cells by a type-III secretion system leading to the subversion of several cellular pathways. However, the molecular function of several effectors remains unknown, even though they contribute to virulence. Here we show that one of them, NleF, binds to caspase-4, -8, and -9 in yeast two-hybrid, LUMIER, and direct interaction assays. NleF inhibits the catalytic activity of the caspases in vitro and in cell lysate and prevents apoptosis in HeLa and Caco-2 cells. We have solved the crystal structure of the caspase-9/NleF complex which shows that NleF uses a novel mode of caspase inhibition, involving the insertion of the carboxy-terminus of NleF into the active site of the protease. In conformance with our structural model, mutagenized NleF with truncated or elongated carboxy-termini revealed a complete loss in caspase binding and apoptosis inhibition. Evasion of apoptosis helps pathogenic E. coli and other pathogens to take over the host cell by counteracting the cell's ability to self-destruct upon infection. Recently, two other effector proteins, namely NleD and NleH, were shown to interfere with apoptosis. Even though NleF is not the only effector protein capable of apoptosis inhibition, direct inhibition of caspases by bacterial effectors has not been reported to date. Also unique so far is its mode of inhibition that resembles the one obtained for synthetic peptide-type inhibitors and as such deviates substantially from previously reported caspase-9 inhibitors such as the BIR3 domain of XIAP

NleF binds caspases -4, -8 and -9.

<p><b>A.</b> Binding of NleF to caspases-4, -8 and -9 using luciferase-NleF and protein-A-caspase fusions (LUMIER assays³⁴). Interaction strengths are expressed as signal to background ratios using binding to protein A as a negative control. The interaction of JUN to FOS serves as a positive control. Squares indicate individual measurements. <b>B,C.</b> The affinity constant (K_d) of NleF binding to immobilized caspase-9 as measured by surface acoustic wave (SAW) technology is ∼39 nM. The K_d is defined as follows: K_d = K_off/K_on = y-intercept/slope (<b>C)</b>, SAW overlay plot of NleF injections. NleF concentrations are indicated in the legend. The two bold curves added to each NleF measurement (on the left and right of the peak) represent the optimal K_obs and K_off curves.</p

Crystal structure of Caspase-9 with bound NleF.

<p><b>A.</b> The caspase-9 protease monomers are shown in magenta and yellow, the active site cysteines (Cys285) are shown as red sticks for improved special orientation of the catalytic site. The NleF effector proteins attached to the caspase active sites are shown in green, as labeled in the figure. <b>B.</b> The C-terminal residues of NleF are anchoring the effector protein to the specificity pockets of the protease. The Fo-Fc omit electron density contoured at 2.5 σ is shown in blue for a representative part of the involved C-terminal NleF residues. <b>C.</b> Detailed binding mode of the NleF C-terminus to the caspase-9 active site. Involved amino acids are shown as green and yellow sticks, hydrogen bonds are depicted as magenta dashed lines. <b>D.</b> Superposition of the NleF(green)-bound caspase-9 (yellow) with the peptide inhibitor Z-EVD-Dcbmk (magenta) bound to the caspase-9 monomer (blue) captured in active state (PDB entry 1JXQ). While the conformation of caspase residues is virtually identical, slight differences in the mode of interaction of the bound inhibitors are e.g. observed for the Asp/Gly-P1 moiety, where the Asp of Z-EVD-Dcbmk adopts a more favourable geometry for efficient salt bridge formation to Arg179. <b>E.</b> Superimposition of the NleF-bound caspase-9 dimer presented in this study and the previously described caspase-9 crystal structure (light blue) in complex with the BIR3 domain of the XIAP inhibitor shown as red cartoon (PDB entry 1NW9). While the latter interferes with the formation of a productive caspase dimer, NleF establishes its inhibitory activity by means of a fundamentally different mechanism of active site targeting. <b>F.</b> Superimposition of the caspase-9 dimer obtained for the NleF-inhibited form (yellow and magenta as in <b>A.,</b> both NleF molecules omitted for clarity) and the dimer of 1JXQ (grey cartoon). While the type of dimer formation and the overall protein conformation is well conserved between the two dimers, deviations arise mainly from the fact that one of the two 1JXQ monomers (left) corresponds to a non-productive, inactive conformation while in the NleF-inhibited dimer, both monomers adopt an active state with well-formed specificity pockets.</p

Caspase binding and apoptosis inhibition by modified versions of NleF.

<p><b>A–C.</b> Caspase-9-binding of different versions of NleF assessed by LUMIER assays. <b>A.</b> Caspase-4, <b>B.</b> caspase-8, <b>C.</b> caspase-9. <b>D.</b> Percentage of apoptotic HeLa cells expressing different versions of NleF after induction with staurosporine. NleF +1: NleF with an additional C-terminal alanine; NleF -1 and NleF -4: NleF with the terminal 1 and 4 amino acids removed, respectively; NleF L186A, NleF Q187A, NleF C188A and NleF G189A: NleF versions with indicated amino acids substituted by alanine; NleF +18: NleF with additional 18 amino acids.</p