Efficient disruption of the function of the mnuA nuclease gene using the endogenous CRISPR/Cas system in Mycoplasma gallisepticum.

Mycoplasmas are important animal pathogens, but the functions and roles of many of their genes in pathogenesis remain unclear, in large part because of the limited tools available for targeted mutagenesis in these bacteria. In this study we used the Mycoplasma gallisepticum CRISPR/Cas system to target a nuclease gene, MGA_0637 (mnuA), which is predicted to play a role in survival and virulence. Our strategy used simultaneous targeting of the ksgA kasugamycin resistance gene, as a mutation in this gene would not interfere with replication but would confer a readily detectable and selectable phenotype in transformants.

Metagenomic investigation of potential abortigenic pathogens in foetal tissues from Australian horses.

Background: Abortion in horses leads to economic and welfare losses to the equine industry. Most cases of equine abortions are sporadic, and the cause is often unknown. This study aimed to detect potential abortigenic pathogens in equine abortion cases in Australia using metagenomic deep sequencing methods.

Characterisation of putative lactate synthetic pathways of Coxiella burnetii.

The zoonotic pathogen Coxiella burnetii, the causative agent of the human disease Q fever, is an ever-present danger to global public health. Investigating novel metabolic pathways necessary for C. burnetii to replicate within its unusual intracellular niche may identify new therapeutic targets.

A Glycerol ABC Transporter Involved in Pathogenicity.

MalF has been shown to be required for virulence in the important avian pathogen To characterize the function of MalF, predicted to be part of a putative ABC transporter, we compared metabolite profiles of a mutant with a transposon inserted in (MalF-deficient ST mutant 04-1; Δ) with those of wild-type bacteria using gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Of the substrates likely to be transported by an ABC transport system, glycerol was detected at significantly lower abundance in the Δ mutant, compared to the wild type. Stable isotope labeling using [U-C]glycerol and reverse transcription-quantitative PCR analysis indicated that MalF was responsible for the import of glycerol into and that, in the absence of MalF, the transcription of , which encodes a second transporter, GtsA, was upregulated, potentially to increase the import of glycerol-3-phosphate into the cell to compensate for the loss of MalF.

A 25-year retrospective study of in association with equine reproductive loss in Australia.

is primarily a pathogen of birds but can also cause disease in other species. Equine reproductive loss caused by has recently been identified in Australia where cases of human disease were also reported in individuals exposed to foetal membranes from an ill neonatal foal in New South Wales. The prevalence of in association with equine reproductive over time and in different regions of Australia is not known.

Mycoplasma bovis Encodes a Novel LRR Lipoprotein That Undergoes Proteolytic Processing and Binds Host Extracellular Matrix Components.

causes serious infections in ruminants, leading to huge economic losses. Lipoproteins are key components of the mycoplasma membrane and are believed to function in nutrient acquisition, adherence, enzymatic interactions with the host, and induction of the host's immune response to infection. Many genes of have not been assigned functions, in part because of their low sequence similarity with other bacteria, making it difficult to extrapolate gene functions.

Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system.

New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen.

EirA Is a Novel Protein Essential for Intracellular Replication of Coxiella burnetii.

The zoonotic bacterial pathogen is the causative agent of Q fever, a febrile illness which can cause a serious chronic infection. is a unique intracellular bacterium which replicates within host lysosome-derived vacuoles. The ability of to replicate within this normally hostile compartment is dependent on the activity of the Dot/Icm type 4B secretion system.

SdrA, an NADP(H)-regenerating enzyme, is crucial for Coxiella burnetii to resist oxidative stress and replicate intracellularly.

Coxiella burnetii, the causative agent of the zoonotic disease Q fever, is a Gram-negative bacterium that replicates inside macrophages within a highly oxidative vacuole. Screening of a transposon mutant library suggested that sdrA, which encodes a putative short-chain dehydrogenase, is required for intracellular replication. Short-chain dehydrogenases are NADP(H)-dependent oxidoreductases, and SdrA contains a predicted NADP binding site, suggesting it may facilitate NADP(H) regeneration by C.

Meso-tartrate inhibits intracellular replication of Coxiella burnetii, the causative agent of the zoonotic disease Q fever.

The zoonotic disease Q fever caused by the intracellular bacterium Coxiella burnetii remains a global health threat due to its high infectivity, environmental stability, the debilitating nature and the long duration of treatment. Designing new and potent drugs that target previously unexplored pathways is essential to shorten treatment time and minimise antibiotic resistance. Nicotinamide adenine dinucleotide (NAD) is an essential and ubiquitous cofactor in all living organisms.

Coxiella burnetii utilizes both glutamate and glucose during infection with glucose uptake mediated by multiple transporters.

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome.

A combined metabolomic and bioinformatic approach to investigate the function of transport proteins of the important pathogen Mycoplasma bovis.

Mycoplasma bovis is an economically important pathogen of the cattle industry worldwide, and there is an urgent need for a more effective vaccine to control the diseases caused by this organism. Although the M. bovis genome sequence is available, very few gene functions of M.

Koala and Wombat Gammaherpesviruses Encode the First Known Viral NTPDase Homologs and Are Phylogenetically Divergent from All Known Gammaherpesviruses.

There is a large taxonomic gap in our understanding of mammalian herpesvirus genetics and evolution corresponding to those herpesviruses that infect marsupials, which diverged from eutherian mammals approximately 150 million years ago (mya). We compare the genomes of two marsupial gammaherpesviruses, (PhaHV1) and (VoHV1), which infect koalas () and wombats (), respectively. The core viral genomes were approximately 117 kbp and 110 kbp in length, respectively, sharing 69% pairwise nucleotide sequence identity.

NAD synthesis is required for intracellular replication of , the causative agent of the neglected zoonotic disease Q fever.

is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that , encoding a putative l-aspartate oxidase required for NAD synthesis, is needed for intracellular replication.

Metabolite profiling of Mycoplasma gallisepticum mutants, combined with bioinformatic analysis, can reveal the likely functions of virulence-associated genes.

Mycoplasma gallisepticum is an economically important pathogen of commercial poultry. An improved understanding of M. gallisepticum pathogenesis is required to develop better control methods.

The major membrane nuclease MnuA degrades neutrophil extracellular traps induced by Mycoplasma bovis.

Mycoplasma bovis has been increasingly recognised worldwide as an economically important pathogen of cattle, causing a range of diseases, including pneumonia, mastitis, polyarthritis and otitis media. It is believed that M. bovis utilises a range of cell surface proteins, including nucleases, to evade the host immune response and survive.

Variation in the microbiome of the urogenital tract of Chlamydia-free female koalas (Phascolarctos cinereus) with and without 'wet bottom'.

Koalas (Phascolarctos cinereus) are iconic Australian marsupials currently threatened by several processes, including infectious diseases and ecological disruption. Infection with Chlamydia pecorum, is considered a key driver of population decline. The clinical sign of 'wet bottom', a staining of the rump associated with urinary incontinence, is often caused by chlamydial urinary tract infections.

Analysis of the Mycoplasma bovis lactate dehydrogenase reveals typical enzymatic activity despite the presence of an atypical catalytic site motif.

The lactate dehydrogenase (LDH) of Mycoplasma genitalium has been predicted to also act as a malate dehydrogenase (MDH), but there has been no experimental validation of this hypothesized dual function for any mollicute. Our analysis of the metabolite profile of Mycoplasma bovis using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) detected malate, suggesting that there may be MDH activity in M. bovis.

Koala retrovirus genotyping analyses reveal a low prevalence of KoRV-A in Victorian koalas and an association with clinical disease.

Purpose: Koala retrovirus (KoRV) is undergoing endogenization into the genome of koalas in Australia, providing an opportunity to assess the effect of retrovirus infection on the health of a population. The prevalence of KoRV in north-eastern Australia (Queensland and New South Wales) is 100 %, whereas previous preliminary investigations in south-eastern Australia (Victoria) suggested KoRV is present at a lower prevalence, although the values have varied widely. Here, we describe a large study of free-ranging koalas in Victoria to estimate the prevalence of KoRV and assess the clinical significance of KoRV infection in wild koalas.

Chlamydia pecorum Infection in Free-ranging Koalas ( Phascolarctos cinereus ) on French Island, Victoria, Australia.

We detected Chlamydia pecorum in two koalas ( Phascolarctos cinereus ) from a closed island population in Victoria, Australia, previously free of Chlamydia infection. The ompA and multilocus sequence type were most closely related to published isolates of livestock rather than koala origin, suggesting potential cross-species transmission of C. pecorum .

Identification of unusual Chlamydia pecorum genotypes in Victorian koalas (Phascolarctos cinereus) and clinical variables associated with infection.

Chlamydia pecorum infection is a threat to the health of free-ranging koalas (Phascolarctos cinereus) in Australia. Utilizing an extensive sample archive we determined the prevalence of C. pecorum in koalas within six regions of Victoria, Australia.

Golgi-located NTPDase1 of Leishmania major is required for lipophosphoglycan elongation and normal lesion development whereas secreted NTPDase2 is dispensable for virulence.

Parasitic protozoa, such as Leishmania species, are thought to express a number of surface and secreted nucleoside triphosphate diphosphohydrolases (NTPDases) which hydrolyze a broad range of nucleoside tri- and diphosphates. However, the functional significance of NTPDases in parasite virulence is poorly defined. The Leishmania major genome was found to contain two putative NTPDases, termed LmNTPDase1 and 2, with predicted NTPDase catalytic domains and either an N-terminal signal sequence and/or transmembrane domain, respectively.

Multiple ecto-nucleoside triphosphate diphosphohydrolases facilitate intracellular replication of Legionella pneumophila.

Legionella pneumophila is an opportunistic pathogen that replicates within alveolar macrophages resulting in the onset of severe atypical pneumonia. Previously we have identified Lpg1905, a eukaryotic-type ecto-NTPDase (nucleoside triphosphate diphosphohydrolase) from L. pneumophila that was required for optimal intracellular replication and virulence in a mouse lung infection model.

Leishmania major methionine sulfoxide reductase A is required for resistance to oxidative stress and efficient replication in macrophages.

Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues.