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.

Staphylococcus pseudintermedius is an opportunistic and emerging zoonotic pathogen that primarily colonises the skin of dogs. Many common variants are methicillin resistant (MRSP) or multidrug resistant (MDR), and drug resistance is increasingly reported across the globe. In New Zealand, MRSP isolation remains rare in clinics. To pre-emptively inform diagnostic and antimicrobial stewardship practices, we examine isolates of S. pseudintermedius, MRSP and MDR-MRSP from New Zealand dogs using a combination of methodologies. Genetic and genomic data combined with antimicrobial susceptibility screening identify common drug-resistance profiles and their genetic determinants. We demonstrate that sensitive and specific species-level identification of S. pseudintermedius can be achieved using Bruker MALDI-TOF MS and, further, that this technique can be used to identify some common subtype variants, providing a level of categorical precision that falls somewhere between single-locus and multi-locus sequence typing. Comparative genomics analysis of global S. pseudintermedius data shows that MRSP moves frequently across the globe, but that horizontal gene transfer events resulting in the acquisition of the SCCmec cassette (responsible for beta-lactam antibiotic resistance) are infrequent. This suggests that biosecurity and surveillance in addition to antibiotic stewardship should play important roles in mitigating the risk of MRSP, especially in countries such as New Zealand where MRSP is still rare.

Enteropathogenic Escherichia coli (EPEC) primarily infects children in developing countries and causes diarrhea that can be deadly. EPEC pathogenesis occurs through type III secretion system (T3SS)-mediated injection of effectors into intestinal epithelial cells (IECs); these effectors alter actin dynamics, modulate the immune response, and disrupt tight junction (TJ) integrity. The resulting compromised barrier function and increased gastrointestinal (GI) permeability may be responsible for the clinical symptoms of infection. Type I interferon (IFN) mediates anti-inflammatory activities and serves essential functions in intestinal immunity and homeostasis; however, its role in the immune response to enteric pathogens, such as EPEC, and its impact on IEC barrier function have not been examined. Here, we report that IFN-β is induced following EPEC infection and regulates IEC TJ proteins to maintain barrier function. The EPEC T3SS effector NleD counteracts this protective activity by inhibiting IFN-β induction and enhancing tumor necrosis factor alpha to promote barrier disruption. The endoribonuclease RNase L is a key mediator of IFN induction and action that promotes TJ protein expression and IEC barrier integrity. EPEC infection inhibits RNase L in a T3SS-dependent manner, providing a mechanism by which EPEC evades IFN-induced antibacterial activities. This work identifies novel roles for IFN-β and RNase L in IEC barrier functions that are targeted by EPEC effectors to escape host defense mechanisms and promote virulence. The IFN-RNase L axis thus represents a potential therapeutic target for enteric infections and GI diseases involving compromised barrier function.

Leptospirosis in New Zealand has been strongly associated with animal-contact occupations and with serovars Hardjo and Pomona. However, recent data suggest changes in these patterns, hence, serovar-specific epidemiology of leptospirosis from 1999 to 2017 was investigated. The 19-year average annual incidence is 2.01/100,000. Early (1999–2007) and late (2008–2017) study period comparisons showed a significant increase in notifications with serovar Ballum (IRR: 1.59, 95% CI: 1.22–2.09) in all cases and serovar Tarassovi (IRR: 1.75, 95% CI: 1.13–2.78) in Europeans and a decrease in notifications with serovars Hardjo and Pomona in all cases. Incidences of Ballum peaked in winter, Hardjo peaked in spring and Tarassovi peaked in summer. Incidence was highest in Māori (2.24/100,000) with dominant serovars being Hardjo and Pomona. Stratification by occupation showed meat workers had the highest incidence of Hardjo (57.29/100,000) and Pomona (45.32/100,000), farmers had the highest incidence of Ballum (11.09/100,000) and dairy farmers had the highest incidence of Tarassovi (12.59/100,000). Spatial analysis showed predominance of Hardjo and Pomona in Hawke’s Bay, Ballum in West Coast and Northland and Tarassovi in Waikato, Taranaki and Northland. This study highlights the serovar-specific heterogeneity of human leptospirosis in New Zealand that should be considered when developing control and prevention strategies.

To identify inhibitors of the essential chromosome partitioning protein ParA that are active against Mycobacterium tuberculosis. Antisense expression of the parA orthologue MSMEG_6939 was induced on the Mycobacterium smegmatis background. Screening of synthetic chemical libraries was performed to identify compounds with higher anti-mycobacterial activity in the presence of parA antisense. Differentially active compounds were validated for specific inhibition of purified ParA protein from M. tuberculosis (Rv3918c). ParA inhibitors were then characterized for their activity towards M. tuberculosis in vitro. Under a number of culture conditions, parA antisense expression in M. smegmatis resulted in reduced growth. This effect on growth provided a basis for the detection of compounds that increased susceptibility to expression of parA antisense. Two compounds identified from library screening, phenoxybenzamine and octoclothepin, also inhibited the in vitro ATPase activity of ParA from M. tuberculosis. Structural in silico analyses predict that phenoxybenzamine and octoclothepin undergo interactions compatible with the active site of ParA. Octoclothepin exhibited significant bacteriostatic activity towards M. tuberculosis. Our data support the use of whole-cell differential antisense screens for the discovery of inhibitors of specific anti-tubercular drug targets. Using this approach, we have identified an inhibitor of purified ParA and whole cells of M. tuberculosis.

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.