The Role and Targets of the RNA-Binding Protein ProQ in the Gram-Negative Bacterial Pathogen Pasteurella multocida.

The Gram-negative pathogen Pasteurella multocida is the causative agent of many important animal diseases. While a number of P. multocida virulence factors have been identified, very little is known about how gene expression and protein production is regulated in this organism. One mechanism by which bacteria regulate transcript abundance and protein production is riboregulation, which involves the interaction of a small RNA (sRNA) with a target mRNA to alter transcript stability and/or translational efficiency. This interaction often requires stabilization by an RNA-binding protein such as ProQ or Hfq. In Escherichia coli and a small number of other species, ProQ has been shown to play a critical role in stabilizing sRNA-mRNA interactions and preferentially binds to the 3' stem-loop regions of the mRNA transcripts, characteristic of intrinsic transcriptional terminators. The aim of this study was to determine the role of ProQ in regulating P. multocida transcript abundance and identify the RNA targets to which it binds. We assessed differentially expressed transcripts in a proQ mutant and identified sites of direct ProQ-RNA interaction using in vivo UV-cross-linking and analysis of cDNA (CRAC). These analyses demonstrated that ProQ binds to, and stabilizes, ProQ-dependent sRNAs and transfer RNAs in P. multocida via adenosine-enriched, highly structured sequences. The binding of ProQ to two RNA molecules was characterized, and these analyses showed that ProQ bound within the coding sequence of the transcript PmVP161_1121, encoding an uncharacterized protein, and within the 3' region of the putative sRNA Prrc13. IMPORTANCE Regulation in P. multocida involving the RNA-binding protein Hfq is required for hyaluronic acid capsule production and virulence. This study further expands our understanding of riboregulation by examining the role of a second RNA-binding protein, ProQ, in transcript regulation and abundance in P. multocida.

Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas).

After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest in green sea turtles (Chelonia mydas), RNA-sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 h after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with upregulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly downregulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including the gene encoding insulin-like growth factor binding protein 1 (igfbp1), were downregulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported to be associated with hypoxia-induced embryonic arrest in diverse taxa, including the nematode Caenorhabditis elegans and zebrafish (Danio rerio). Hypoxia-induced preovipositional embryonic arrest appears to be a unique adaptation of turtles. However, our findings accord with the proposition that the mechanisms underlying hypoxia-induced embryonic arrest per se are highly conserved across diverse taxa.

Characterization of Hypermutator Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis in Australia.

Hypermutable Pseudomonas aeruginosa isolates (hypermutators) have been identified in patients with cystic fibrosis (CF) and are associated with reduced lung function. Hypermutators display a greatly increased mutation rate and an enhanced ability to become resistant to antibiotics during treatment. Their prevalence has been established among patients with CF, but it has not been determined for patients with CF in Australia. This study aimed to determine the prevalence of hypermutable P. aeruginosa isolates from adult patients with CF from a health care institution in Australia and to characterize the genetic diversity and antibiotic susceptibility of these isolates. A total of 59 P. aeruginosa clinical isolates from patients with CF were characterized. For all isolates, rifampin (RIF) mutation frequencies and susceptibility to a range of antibiotics were determined. Of the 59 isolates, 13 (22%) were hypermutable. Whole-genome sequences were determined for all hypermutable isolates. Core genome polymorphisms were used to assess genetic relatedness of the isolates, both to each other and to a sample of previously characterized P. aeruginosa strains. Phylogenetic analyses showed that the hypermutators were from divergent lineages and that hypermutator phenotype was mostly the result of mutations in mutL or, less commonly, in mutS Hypermutable isolates also contained a range of mutations that are likely associated with adaptation of P. aeruginosa to the CF lung environment. Multidrug resistance was more prevalent in hypermutable than nonhypermutable isolates (38% versus 22%). This study revealed that hypermutable P. aeruginosa strains are common among isolates from patients with CF in Australia and are implicated in the emergence of antibiotic resistance.

Comparable Efficacy and Better Safety of Double ß-Lactam Combination Therapy versus ß­Lactam plus Aminoglycoside in Gram-Negative Bacteria in Randomized, Controlled Trials.

There is a great need for efficacious therapies against Gram-negative bacteria. Double ß-lactam combination(s) (DBL) are relatively safe, and preclinical data are promising; however, their clinical role has not been well defined. We conducted a metaanalysis of the clinical and microbiological efficacy of DBL compared to ß-lactam plus aminoglycoside combinations (BLAG). PubMed, Embase, ISI Web of Knowledge, and Cochrane Controlled Trials Register database were searched through July 2018. We included randomized controlled clinical trials that compared DBL with BLAG combinations. Clinical response was used as the primary outcome and microbiological response in Gram-negative bacteria as the secondary outcome; sensitivity analyses were performed for Pseudomonas aeruginosa, Klebsiella spp., and Escherichia coli Heterogeneity and risk of bias were assessed. Safety results were classified by systems and organs. Thirteen studies evaluated 2,771 cases for clinical response and 665 cases for microbiological response in various Gram-negative species. DBL achieved slightly, but not significantly, better clinical response (risk ratio, 1.05; 95% confidence interval [CI], 0.99 to 1.11) and microbiological response in Gram-negatives (risk ratio, 1.11; 95% CI, 0.99 to 1.25) compared with BLAG. Sensitivity analyses by pathogen showed the same trend. No significant heterogeneity across studies was found. DBL was significantly safer than BLAG regarding renal toxicity (6.6% versus 8.8%, P = 0.0338) and ototoxicity (0.7 versus 3.1%, P = 0.0137). Other adverse events were largely comparable. Overall, empirically designed DBL showed comparable clinical and microbiological responses across different Gram-negative species, and were significantly safer than BLAG. Therefore, DBL should be rationally optimized via the latest translational approaches, leveraging mechanistic insights and newer ß-lactams for future evaluation in clinical trials.

Emergence of High-Level Colistin Resistance in an Acinetobacter baumannii Clinical Isolate Mediated by Inactivation of the Global Regulator H-NS.

Colistin is a crucial last-line drug used for the treatment of life-threatening infections caused by multidrug-resistant strains of the Gram-negative bacterium Acinetobacter baumannii However, colistin-resistant A. baumannii isolates can still be isolated following failed colistin therapy. Resistance is most often mediated by the addition of phosphoethanolamine (pEtN) to lipid A by PmrC, following missense mutations in the pmrCAB operon encoding PmrC and the two-component signal transduction system PmrA/PmrB. We recovered a pair of A. baumannii isolates from a single patient before (6009-1) and after (6009-2) failed colistin treatment. These strains displayed low and very high levels of colistin resistance (MICs, 8 to 16 µg/ml and 128 µg/ml), respectively. To understand how increased colistin resistance arose, we sequenced the genome of each isolate, which revealed that 6009-2 had an extra copy of the insertion sequence element ISAba125 within a gene encoding an H-NS family transcriptional regulator. To confirm the role of H-NS in colistin resistance, we generated an hns deletion mutant in 6009-1 and showed that colistin resistance increased upon the deletion of hns We also provided 6009-2 with an intact copy of hns and showed that the strain was no longer resistant to high concentrations of colistin. Transcriptomic analysis of the clinical isolates identified more than 150 genes as being differentially expressed in the colistin-resistant hns mutant 6009-2. Importantly, the expression of eptA, encoding a second lipid A-specific pEtN transferase but not pmrC, was increased in the hns mutant. This is the first time an H-NS family transcriptional regulator has been associated with a pEtN transferase and colistin resistance.

Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2.

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.

Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS. RESULTS: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone. CONCLUSIONS: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes.

The transcriptomic response of Acinetobacter baumannii to colistin and doripenem alone and in combination in an in vitro pharmacokinetics/pharmacodynamics model.

OBJECTIVES: Colistin remains a last-line treatment for MDR Acinetobacter baumannii and combined use of colistin and carbapenems has shown synergistic effects against MDR strains. In order to understand the bacterial responses to these antibiotics, we analysed the transcriptome of A. baumannii following exposure to each. METHODS: RNA sequencing was employed to determine changes in the transcriptome following treatment with colistin and doripenem, both alone and in combination, using an in vitro pharmacokinetics (PK)/pharmacodynamics model to mimic the PK of both antibiotics in patients. RESULTS: After treatment with colistin (continuous infusion at 2 mg/L), >400 differentially regulated genes were identified, including many associated with outer membrane biogenesis, fatty acid metabolism and phospholipid trafficking. No genes were differentially expressed following treatment with doripenem (Cmax 25 mg/L, t1/2 1.5 h) for 15 min, but 45 genes were identified as differentially expressed after 1 h of growth under this condition. Treatment of A. baumannii with both colistin and doripenem together for 1 h resulted in >450 genes being identified as differentially expressed. More than 70% of these gene expression changes were also observed following colistin treatment alone. CONCLUSIONS: These data suggest that colistin causes gross damage to the outer membrane, facilitates lipid exchange between the inner and outer membrane and alters the normal asymmetric outer membrane composition. The transcriptional response to colistin was highly similar to that observed for an LPS-deficient strain, indicating that many of the observed changes are responses to outer membrane instability resulting from LPS loss.

The C-terminal protein interaction domain of the chromatin reader Yaf9 is critical for pathogenesis of Candida albicans.

Fungal infections cause a large health burden but are treated by only a handful of antifungal drug classes. Chromatin factors have emerged as possible targets for new antifungals. These targets include the reader proteins, which interact with posttranslationally modified histones to influence DNA transcription and repair. The YEATS domain is one such reader recognizing both crotonylated and acetylated histones. Here, we performed a detailed structure/function analysis of the Candida albicans YEATS domain reader Yaf9, a subunit of the NuA4 histone acetyltransferase and the SWR1 chromatin remodeling complex. We have previously demonstrated that the homozygous deletion mutant yaf9Δ/Δ displays growth defects and is avirulent in mice. Here we show that a YEATS domain mutant expected to inactivate Yaf9's chromatin binding does not display strong phenotypes in vitro, nor during infection of immune cells or in a mouse systemic infection model, with only a minor virulence reduction in vivo. In contrast to the YEATS domain mutation, deletion of the C-terminal domain of Yaf9, a protein-protein interaction module necessary for its interactions with SWR1 and NuA4, phenocopies the null mutant. This shows that the C-terminal domain is essential for Yaf9 roles in vitro and in vivo, including C. albicans virulence. Our study informs on the strategies for therapeutic targeting of Yaf9, showing that approaches taken for the mammalian YEATS domains by disrupting their chromatin binding might not be effective in C. albicans, and provides a foundation for studying YEATS proteins in human fungal pathogens.IMPORTANCEThe scarcity of available antifungal drugs and rising resistance demand the development of therapies with new modes of action. In this context, chromatin regulation may be a target for novel antifungal therapeutics. To realize this potential, we must better understand the roles of chromatin regulators in fungal pathogens. Toward this goal, here, we studied the YEATS domain chromatin reader Yaf9 in Candida albicans. Yaf9 uses the YEATS domain for chromatin binding and a C-terminal domain to interact with chromatin remodeling complexes. By constructing mutants in these domains and characterizing their phenotypes, our data indicate that the Yaf9 YEATS domain might not be a suitable therapeutic drug target. Instead, the Yaf9 C-terminal domain is critical for C. albicans virulence. Collectively, our study informs how a class of chromatin regulators performs their cellular and pathogenesis roles in C. albicans and reveals strategies to inhibit them.

Combating Multidrug-Resistant Bacteria by Integrating a Novel Target Site Penetration and Receptor Binding Assay Platform Into Translational Modeling.

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of ß-lactam antibiotics and ß-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While ß-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.

Disruption of the Burkholderia pseudomallei two-component signal transduction system BbeR-BbeS leads to increased extracellular DNA secretion and altered biofilm formation.

Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS.

Novel Cassette Assay To Quantify the Outer Membrane Permeability of Five ß-Lactams Simultaneously in Carbapenem-Resistant Klebsiella pneumoniae and Enterobacter cloacae.

Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While ß-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a blaKPC-2 and several other ß-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular ß-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our ß-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple ß-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of ß-lactam antibiotics.IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While ß-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of ß-lactams leads to high drug concentrations at their periplasmic target sites, allowing ß-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of ß-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five ß-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem ß-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of ß-lactams and combat carbapenem-resistant isolates.

Systematic Identification and Analysis of Acinetobacter baumannii Type VI Secretion System Effector and Immunity Components.

Many Gram-negative bacteria use a type VI secretion system (T6SS) for microbial warfare and/or host manipulation. Acinetobacter baumannii is an important nosocomial pathogen and many A. baumannii strains utilize a T6SS to deliver toxic effector proteins to surrounding bacterial cells. These toxic effectors are usually delivered together with VgrG proteins, which form part of the T6SS tip complex. All previously identified A. baumannii T6SS effectors are encoded within a three- or four-gene locus that also encodes a cognate VgrG and immunity protein, and sometimes a chaperone. In order to characterize the diversity and distribution of T6SS effectors and immunity proteins in this species, we first identified all vgrG genes in 97 A. baumannii strains via the presence of the highly conserved VgrG domain. Most strains encoded between two and four different VgrG proteins. We then analyzed the regions downstream of the identified vgrG genes and identified more than 240 putative effectors. The presence of conserved domains in these effectors suggested a range of functions, including peptidoglycan hydrolases, lipases, nucleases, and nucleic acid deaminases. However, 10 of the effector groups had no functionally characterized domains. Phylogenetic analysis of these putative effectors revealed that they clustered into 32 distinct groups that appear to have been acquired from a diverse set of ancestors. Corresponding immunity proteins were identified for all but two of the effector groups. Effectors from eight of the 32 groups contained N-terminal rearrangement hotspot (RHS) domains. The C-terminal regions of these RHS proteins, which are predicted to confer the toxic effector function, were very diverse, but the N-terminal RHS domains clustered into just two groups. While the majority of A. baumannii strains contained an RHS type effector, no strains encoded two RHS effectors with similar N-terminal sequences, suggesting that the presence of similar N-terminal RHS domains leads to competitive exclusion. Together, these analyses define the extreme diversity of T6SS effectors within A. baumannii and, as many have unknown functions, future detailed characterization of these effectors may lead to the identification of proteins with novel antibacterial properties.

Recombinant LipL32 stimulates interferon-gamma production in cattle vaccinated with a monovalent Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis vaccine.

Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) is the main causative agent of bovine leptospirosis in Australia, New Zealand, North America and elsewhere. Bovine leptospirosis can result in spontaneous abortion, stillbirth and reduced milk output. The organism is shed in the urine of infected animals and contact with contaminated materials can result in zoonotic infections in humans. Protective immunity in cattle against Hardjobovis involves stimulation of a Th1 cell mediated immune response, which can be characterized by the production of IFN-γ when blood from vaccinated animals is exposed to Hardjobovis antigens. However, the leptospiral components involved in stimulating this response have yet to be identified. In this study, 238 recombinant leptospiral proteins were evaluated for their ability to stimulate IFN-γ production in blood of cattle vaccinated with a commercial monovalent Hardjobovis vaccine. The conserved lipoprotein LipL32 is the major outer membrane protein of pathogenic Leptospira spp. A pool of soluble recombinant proteins which included LipL32, as well as LipL32 alone, stimulated significant IFN-γ production in blood of vaccinated cattle. A number of recombinant LipL32 fragments was generated, which identified the amino acids between 20 and 200 as containing the bovine T-cell reactive regions of LipL32. However, whether LipL32 plays a role in stimulating protective immunity in mammals has yet to be conclusively determined.