Ticks (Acari: Ixodidae) infesting cattle in coastal Kenya harbor a diverse array of tick-borne pathogens.

Ticks and the microbes they transmit have emerged in sub-Saharan Africa as a major threat to veterinary and public health. Although progress has been made in detecting and identifying tick-borne pathogens (TBPs) across vast agroecologies of Kenya, comprehensive information on tick species infesting cattle and their associated pathogens in coastal Kenya needs to be updated and expanded. Ticks infesting extensively grazed zebu cattle in 14 villages were sampled and identified based on morphology and molecular methods and tested for the presence of bacterial and protozoan TBPs using PCR with high-resolution melting analysis and gene sequencing. In total, 3,213 adult ticks were collected and identified as Rhipicephalus appendiculatus (15.8%), R. evertsi (12.8%), R. microplus (11.3%), R. pulchellus (0.1%), Amblyomma gemma (24.1%), A. variegatum (35.1%), Hyalomma rufipes (0.6%), and H. albiparmatum (0.2%). Ticks were infected with Rickettsia africae, Ehrlichia ruminantium, E. minasensis, Theileria velifera and T. parva. Coxiella sp. endosymbionts were detected in the Rhipicephalus and Amblyomma ticks. Co-infections with two and three different pathogens were identified in 6.9% (n = 95/1382) and 0.1% (n = 2/1382) of single tick samples, respectively, with the most common co-infection being R. africae and E. ruminantium (7.2%, CI: 4.6 - 10.6). All samples were negative for Coxiella burnetii, Anaplasma spp. and Babesia spp. Our study provides an overview of tick and tick-borne microbial diversities in coastal Kenya.

Epidemiology of tick-borne pathogens of cattle and tick control practices in coastal Kenya.

Tick-borne diseases (TBD) are a major constraint to livestock health and productivity in sub-Saharan Africa. Nonetheless, there are relatively few robust epidemiologic studies documenting TBD and its management in different endemic settings in Kenya. Therefore, a cross-sectional study using multi-stage cluster sampling was undertaken to characterize the epidemiology of TBD and management factors among zebu cattle reared under an extensive system in coastal Kenya. Blood samples from 1486 cattle from 160 herds in 14 villages were screened for the presence of tick-borne bacterial and protozoan pathogens using PCR with high-resolution melting analysis and sequencing. Standardized questionnaires were used to collect data on herd structure and herd management practices, and a mixed-effect logistic regression model to identify risk factors for tick-borne pathogens (TBPs). The application of chemical acaricide was the primary method for tick control (96.3%, 154/160), with the amidine group (mainly Triatix®, amitraz) being the most frequently used acaricides. Respondents identified East Coast fever as the most important disease and Butalex® (buparvaquone) was the most commonly administered drug in response to perceived TBD in cattle. The overall animal- and herd-level prevalence for TBPs were 24.2% (95% confidence interval (CI): 22.0-26.4%) and 75.6% (95% CI: 68.2-82.1%), respectively. Cattle were infected with Anaplasma marginale (10.9%, 95% CI: 9.4-12.6), Theileria parva (9.0%, 95% CI: 7.5-10.5), Anaplasma platys (2.6%, 95% CI: 1.9-3.6), Theileria velifera (1.1%, 95% CI: 0.7-1.8), Babesia bigemina (0.5%, 95% CI: 0.2-1.0), and Anaplasma sp. (0.1%, 95% CI: 0.0-0.4). Moreover, 21 cattle (1.4%) were co-infected with two TBPs. None of the assessed potential risk factors for the occurrence of either A. marginale or T. parva in cattle were statistically significant. The intra-herd correlation coefficients (lCCs) computed in this study were 0.29 (A. marginale) and 0.14 (T. parva). This study provides updated molecular-based information on the epidemiological status of TBPs of cattle and herd management practices in coastal Kenya. This information can be used in designing cost-effective control strategies for combating these TBD in the region.

Evaluating the efficacy of Mazao Tickoff (Metarhizium anisopliae ICIPE 7) in controlling natural tick infestations on cattle in coastal Kenya: Study protocol for a randomized controlled trial.

Ticks and tick-borne diseases cause substantial economic losses to the livestock industry in sub-Saharan Africa. Mazao Tickoff is a novel bioacaricide developed for tick control and is based on the entomopathogenic fungus Metarhizium anisopliae sensu lato (s.l.) isolate ICIPE 7. To date, no randomized controlled study has been undertaken to demonstrate the efficacy of this bioacaricide in reducing natural tick infestation on cattle. To this end, this field trial is designed to evaluate the anti-tick efficacy of Mazao Tickoff on cattle in coastal Kenya compared to a standard chemical tick control protocol. In this prospective, multi-center randomized controlled trial, eligible herds will be randomized by the herd size to the intervention arm in a 1:1:1 ratio to either Triatix® (active ingredient: amitraz); Mazao Tickoff (active ingredient: M. anisopliae ICIPE 7); or placebo (excipients of the Mazao Tickoff), with a total enrollment target of 1,077 cattle. Treatments will be dispensed on Day 0 (defined individually as the day each animal receives the first treatment) and thereafter every two weeks until Day 182. Ticks will be counted on every animal in each herd (herds to be included have at least one animal bearing at least one tick on Day 0), and thereafter on bi-weekly intervals until Day 182. The primary efficacy assessments of Mazao Tickoff will be based on the mean percentage reduction in tick counts at each post-treatment follow-up visit compared to the placebo group and the Triatix® arm. Further, the effect of Mazao Tickoff on the prevalence of common cattle pathogens, Anaplasma marginale and Theileria parva, will be determined by assessing incidence and seroprevalence at four different time points. This protocol describes the first rigorous evaluation of the efficacy of Mazao Tickoff and its potential as a viable alternative non-chemical acaricide tool for tick control in Kenya and elsewhere.

Proteome Profiling of Mycobacterium tuberculosis Cells Exposed to Nitrosative Stress.

Reactive nitrogen species (RNS) are secreted by human cells in response to infection by Mycobacterium tuberculosis (Mtb). Although RNS can kill Mtb under some circumstances, Mtb can adapt and survive in the presence of RNS by a process that involves modulation of gene expression. Previous studies focused primarily on stress-related changes in the Mtb transcriptome. This study unveils changes in the Mtb proteome in response to a sub-lethal dose of nitric oxide (NO) over several hours of exposure. Proteins were identified using liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS/MS). A total of 2911 Mtb proteins were identified, of which 581 were differentially abundant (DA) after exposure to NO in at least one of the four time points (30 min, 2 h, 6 h, and 20 h). The proteomic response to NO was marked by two phases, with few DA proteins in the early phase and a multitude of DA proteins in the later phase. The efflux pump Rv1687 stood out as being the only protein more abundant at all the time points and might play a role in the early protection of Mtb against nitrosative stress. These changes appeared to be compensatory in nature, contributing to iron homeostasis, energy metabolism, and other stress responses. This study thereby provides new insights into the response of Mtb to NO at the level of proteomics.

Anti-Tick Vaccines: Current Advances and Future Prospects.

Ticks are increasingly a global public health and veterinary concern. They transmit numerous pathogens that are of veterinary and public health importance. Acaricides, livestock breeding for tick resistance, tick handpicking, pasture spelling, and anti-tick vaccines (ATVs) are in use for the control of ticks and tick-borne diseases (TTBDs); acaricides and ATVs being the most and least used TTBD control methods respectively. The overuse and misuse of acaricides has inadvertently selected for tick strains that are resistant to acaricides. Furthermore, vaccines are rare and not commercially available in sub-Saharan Africa (SSA). It doesn't help that many of the other methods are labor-intensive and found impractical especially for larger farm operations. The success of TTBD control is therefore dependent on integrating all the currently available methods. Vaccines have been shown to be cheap and effective. However, their large-scale deployment for TTBD control in SSA is hindered by commercial unavailability of efficacious anti-tick vaccines against sub-Saharan African tick strains. Thanks to advances in genomics, transcriptomics, and proteomics technologies, many promising anti-tick vaccine antigens (ATVA) have been identified. However, few of them have been investigated for their potential as ATV candidates. Reverse vaccinology (RV) can be leveraged to accelerate ATV discovery. It is cheap and shortens the lead time from ATVA discovery to vaccine production. This chapter provides a brief overview of recent advances in ATV development, ATVs, ATV effector mechanisms, and anti-tick RV. Additionally, it provides a detailed outline of vaccine antigen selection and analysis using computational methods.

The developmentally dynamic microRNA transcriptome of Glossina pallidipes tsetse flies, vectors of animal trypanosomiasis.

Summary: MicroRNAs (miRNAs) are single stranded gene regulators of 18-25 bp in length. They play a crucial role in regulating several biological processes in insects. However, the functions of miRNA in Glossina pallidipes, one of the biological vectors of African animal trypanosomosis in sub-Saharan Africa, remain poorly characterized. We used a combination of both molecular biology and bioinformatics techniques to identify miRNA genes at different developmental stages (larvae, pupae, teneral and reproductive unmated adults, gravid females) and sexes of G. pallidipes. We identified 157 mature miRNA genes, including 12 novel miRNAs unique to G. pallidipes. Moreover, we identified 93 miRNA genes that were differentially expressed by sex and/or in specific developmental stages. By combining both miRanda and RNAhybrid algorithms, we identified 5550 of their target genes. Further analyses with the Gene Ontology term and KEGG pathways for these predicted target genes suggested that the miRNAs may be involved in key developmental biological processes. Our results provide the first repository of G. pallidipes miRNAs across developmental stages, some of which appear to play crucial roles in tsetse fly development. Hence, our findings provide a better understanding of tsetse biology and a baseline for exploring miRNA genes in tsetse flies. Availability and implementation: Raw sequence data are available from NCBI Sequence Read Archives (SRA) under Bioproject accession number PRJNA590626. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Tsetse Bloodmeal Analyses Incriminate the Common Warthog Phacochoerus africanus as an Important Cryptic Host of Animal Trypanosomes in Smallholder Cattle Farming Communities in Shimba Hills, Kenya.

Trypanosomes are endemic and retard cattle health in Shimba Hills, Kenya. Wildlife in the area act as reservoirs of the parasites. However, wild animal species that harbor and expose cattle to tsetse-borne trypanosomes are not well known in Shimba Hills. Using xeno-monitoring surveillance to investigate wild animal reservoirs and sources of trypanosomes in Shimba Hills, we screened 696 trypanosome-infected and uninfected tsetse flies for vertebrate DNA using multiple-gene PCR-High Resolution Melting analysis and amplicon sequencing. Results revealed that tsetse flies fed on 13 mammalian species, preferentially Phacochoerus africanus (warthogs) (17.39%, 95% CI: 14.56-20.21) and Bos taurus (cattle) (11.35%, 95% CI: 8.99-13.71). Some tsetse flies showed positive cases of bloodmeals from multiple hosts (3.45%, 95% CI: 2.09-4.81), including warthog and cattle (0.57%, 95% CI: 0.01-1.14). Importantly, tsetse flies that took bloodmeals from warthog had significant risk of infections with Trypanosoma vivax (5.79%, 95% CI: 1.57-10.00), T. congolense (7.44%, 95% CI: 2.70-12.18), and T. brucei sl (2.48%, 95% CI: -0.33-5.29). These findings implicate warthogs as important reservoirs of tsetse-borne trypanosomes affecting cattle in Shimba Hills and provide valuable epidemiological insights to underpin the parasites targeted management in Nagana vector control programs in the area.

Prevalence of Trypanosome Species in Cattle Near Ruma National Park, Lambwe Valley, Kenya: An Update From the Historical Focus for African Trypanosomosis.

The effective control of diseases in areas shared with wildlife depends on the validity of the epidemiologic parameters that guide interventions. Epidemiologic data on animal trypanosomosis in Lambwe valley are decades old, and the recent suspected outbreaks of the disease in the valley necessitate the urgent bridging of this data gap. This cross-sectional study estimated the prevalence of bovine trypanosomosis, identified risk factors, and investigated the occurrence of species with zoonotic potential in Lambwe valley. The area is ~324 km2, of which 120 km2 is the Ruma National Park. Blood was sampled from the jugular and marginal ear veins of 952 zebu cattle between December 2018 and February 2019 and tested for trypanosomes using the Buffy Coat Technique (BCT) and PCR-High-Resolution Melting (HRM) analysis of the 18S RNA locus. Risk factors for the disease were determined using logistic regression. The overall trypanosome prevalence was 11.0% by BCT [95% confidence interval (CI): 9.0-13.0] and 27.9% by PCR-HRM (95% CI: 25.1-30.8). With PCR-HRM as a reference, four species of trypanosomes were detected at prevalences of 12.7% for T. congolense savannah (95% CI: 10.6-14.8), 7.7% for T. brucei brucei (CI: 6.0-9.4), 8.7% for T. vivax (CI: 6.9-10.5), and 1.3% for T. theileri (CI: 0.6-2.0). About 2.4% of cattle had mixed infections (CI: 1.4-3.41). No human-infective trypanosomes were found. Infections clustered across villages but were not associated with animal age, sex, herd size, and distance from the park. Approximately 85% of infections occurred within 2 km of the park. These findings add to evidence that previous interventions eliminated human trypanosomosis but not bovine trypanosomosis. Risk-tailored intervention within 2 km of Ruma Park, especially in the north and south ends, coupled with stringent screening with molecular tools, could significantly reduce bovine trypanosomosis.

Molecular characterization of Trypanosoma vivax in tsetse flies confirms the presence of the virulent Tvv4 genotype in Kenya: Potential implications for the control of trypanosomiasis in Shimba Hills.

Trypanosoma vivax is a vector-borne protozoan parasite of livestock endemic to Africa and South America. To date, fifteen genotypes of the parasite have been described in vertebrate and insect hosts in East Africa. However, information regarding T. vivax diversity remains limited in many endemic countries in the sub-region, including Kenya. Such information could deepen insight into the local epidemiology of animal trypanosomiasis in Shimba Hills, a wildlife area in southeast Kenya where T. vivax is endemic and infects livestock. We employed two-gene conventional-PCR-sequencing and phylogenetic analysis to characterize T. vivax genotypes in tsetse flies collected between November 2018 and September 2019 in the wildlife-livestock interface of the Shimba Hills National Reserve. Phylogenetic analysis of Internal Transcribed Spacer-1 (ITS-1) sequences of T. vivax isolates confirmed the presence of two T. vivax genotypes in Shimba Hills of which >80% of T. vivax isolates from tsetse flies clustered within the virulent Tvv4-genotype clade. Tsetse infections with the Tvv4 genotype were also confirmed based on 18S rRNA gene sequencing. Expanded gene characterization identified three closely related haplotypes within the Tvv4-clade. The Tvv4-isolates were detected in male and female Glossina pallidipes tsetse flies, most of which were collected from grasslands and within two kilometres of the Shimba Hills National Reserve boundary. Considering that T. vivax is the most common trypanosome in the Shimba Hills area and causes severe clinical conditions in livestock, the Tvv4 genotype reported here for the first time in Kenya contributes to our understanding of these pathologies. The effectiveness of trypanocidal drugs in the management of Tvv4 is presently not clearly understood. Therefore, the parasite management in Shimba Hills should focus on vector control to reduce the density of G. pallidipes, especially in grasslands near the wildlife protectorate.

Anaplasma and Theileria Pathogens in Cattle of Lambwe Valley, Kenya: A Case for Pro-Active Surveillance in the Wildlife-Livestock Interface.

Tick-borne pathogens (TBPs) are major constraints to livestock production and a threat to public health in Africa. This cross-sectional study investigated the risk of infection with TBPs in cattle of Lambwe Valley, Kenya. Blood samples of 680 zebu cattle from 95 herds in six geospatial clusters within 5 km of Ruma National Park were screened for bacterial and protozoan TBPs by high-resolution melting analysis and sequencing of PCR products. We detected Anaplasma bovis (17.4%), Anaplasma platys (16.9%), Anaplasma marginale (0.6%), Theileria velifera (40%), and Theileria mutans (25.7%), as well as an Anaplasma sp. (11.6%) that matched recently reported Anaplasma sp. sequences from Ethiopia. Babesia, Rickettsia, and Ehrlichia spp. were not detected. The animal and herd-level prevalences for TBPs were 78.5% (95% confidence intervals (CI): 75.3, 81.5) and 95.8% (95% CI: 91.8, 99.8), respectively. About 31.6% of cattle were co-infected with 13 combinations of TBPs. The prevalence of TBPs differed between clusters and age, but the risk of infection was not associated with sex, herd size, or the distance of homesteads from Ruma. This study adds insight into the epidemiology of TBPs around Ruma and highlights the need for proactive surveillance of TBPs in livestock-wildlife interfaces.

Lineage-Specific Proteomic Signatures in the Mycobacterium tuberculosis Complex Reveal Differential Abundance of Proteins Involved in Virulence, DNA Repair, CRISPR-Cas, Bioenergetics and Lipid Metabolism.

Despite the discovery of the tubercle bacillus more than 130 years ago, its physiology and the mechanisms of virulence are still not fully understood. A comprehensive analysis of the proteomes of members of the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages 3, 4, 5, and 7 was conducted to better understand the evolution of virulence and other physiological characteristics. Unique and shared proteomic signatures in these modern, pre-modern and ancient MTBC lineages, as deduced from quantitative bioinformatics analyses of high-resolution mass spectrometry data, were delineated. The main proteomic findings were verified by using immunoblotting. In addition, analysis of multiple genome alignment of members of the same lineages was performed. Label-free peptide quantification of whole cells from MTBC lineages 3, 4, 5, and 7 yielded a total of 38,346 unique peptides derived from 3092 proteins, representing 77% coverage of the predicted proteome. MTBC lineage-specific differential expression was observed for 539 proteins. Lineage 7 exhibited a markedly reduced abundance of proteins involved in DNA repair, type VII ESX-3 and ESX-1 secretion systems, lipid metabolism and inorganic phosphate uptake, and an increased abundance of proteins involved in alternative pathways of the TCA cycle and the CRISPR-Cas system as compared to the other lineages. Lineages 3 and 4 exhibited a higher abundance of proteins involved in virulence, DNA repair, drug resistance and other metabolic pathways. The high throughput analysis of the MTBC proteome by super-resolution mass spectrometry provided an insight into the differential expression of proteins between MTBC lineages 3, 4, 5, and 7 that may explain the slow growth and reduced virulence, metabolic flexibility, and the ability to survive under adverse growth conditions of lineage 7.

Ample glycosylation in membrane and cell envelope proteins may explain the phenotypic diversity and virulence in the Mycobacterium tuberculosis complex.

Multiple regulatory mechanisms including post-translational modifications (PTMs) confer complexity to the simpler genomes and proteomes of Mycobacterium tuberculosis (Mtb). PTMs such as glycosylation play a significant role in Mtb adaptive processes. The glycoproteomic patterns of clinical isolates of the Mycobacterium tuberculosis complex (MTBC) representing the lineages 3, 4, 5 and 7 were characterized by mass spectrometry. A total of 2944 glycosylation events were discovered in 1325 proteins. This data set represents the highest number of glycosylated proteins identified in Mtb to date. O-glycosylation constituted 83% of the events identified, while 17% of the sites were N-glycosylated. This is the first report on N-linked protein glycosylation in Mtb and in Gram-positive bacteria. Collectively, the bulk of Mtb glycoproteins are involved in cell envelope biosynthesis, fatty acid and lipid metabolism, two-component systems, and pathogen-host interaction that are either surface exposed or located in the cell wall. Quantitative glycoproteomic analysis revealed that 101 sites on 67 proteins involved in Mtb fitness and survival were differentially glycosylated between the four lineages, among which 64% were cell envelope and membrane proteins. The differential glycosylation pattern may contribute to phenotypic variabilities across Mtb lineages. The study identified several clinically important membrane-associated glycolipoproteins that are relevant for diagnostics as well as for drug and vaccine discovery.

Expression of nucleotide excision repair in Alzheimer's disease is higher in brain tissue than in blood.

Age-related changes are increased in patients with Alzheimer's disease (AD), including oxidative stress and DNA damage. We propose that genotoxic stress and DNA repair responses influence neurodegeneration in the pathogenesis of AD. Here, we focus on nucleotide excision repair (NER). Real-time qPCR and mass spectrometry were employed to determine the expression levels of selected NER components. The mRNA levels of the genes encoding the NER proteins RAD23B, RPA1, ERCC1, PCNA and LIG3 as well as the NER-interacting base excision repair protein MPG in blood and brain tissue from four brain regions in patients with AD or mild cognitive impairment and healthy controls (HC), were assessed. NER mRNA levels were significantly higher in brain tissue than in blood. Further, LIG3 mRNA levels in the frontal cortex was higher in AD versus HC, while mRNA levels of MPG and LIG3 in entorhinal cortex and RPA1 in the cerebellum were lower in AD versus HC. In blood, RPA1 and ERCC1 mRNA levels were lower in AD patients than in HC. Alterations in gene expression of NER components between brain regions were associated with AD, connecting DNA repair to AD pathogenesis and suggesting a distinct role for NER in the brain.

The helicase DinG responds to stress due to DNA double strand breaks.

Neisseria meningitidis (Nm) is a Gram-negative nasopharyngeal commensal that can cause septicaemia and meningitis. The neisserial DNA damage-inducible protein DinG is a helicase related to the mammalian helicases XPD and FANCJ. These helicases belong to superfamily 2, are ATP dependent and exert 5' → 3' directionality. To better understand the role of DinG in neisserial genome maintenance, the Nm DinG (DinGNm) enzymatic activities were assessed in vitro and phenotypical characterization of a dinG null mutant (NmΔdinG) was performed. Like its homologues, DinGNm possesses 5' → 3' directionality and prefers DNA substrates containing a 5'-overhang. ATPase activity of DinGNm is strictly DNA-dependent and DNA unwinding activity requires nucleoside triphosphate and divalent metal cations. DinGNm directly binds SSBNm with a Kd of 313 nM. Genotoxic stress analysis demonstrated that NmΔdinG was more sensitive to double-strand DNA breaks (DSB) induced by mitomycin C (MMC) than the Nm wildtype, defining the role of neisserial DinG in DSB repair. Notably, when NmΔdinG cells grown under MMC stress assessed by quantitative mass spectrometry, 134 proteins were shown to be differentially abundant (DA) compared to unstressed NmΔdinG cells. Among the DNA replication, repair and recombination proteins affected, polymerase III subunits and recombinational repair proteins RuvA, RuvB, RecB and RecD were significantly down regulated while TopA and SSB were upregulated under stress condition. Most of the other DA proteins detected are involved in metabolic functions. The present study shows that the helicase DinG is probably involved in regulating metabolic pathways as well as in genome maintenance.

Comparative Proteomic Analysis of Mycobacterium tuberculosis Lineage 7 and Lineage 4 Strains Reveals Differentially Abundant Proteins Linked to Slow Growth and Virulence.

In order to decipher the nature of the slowly growing Mycobacterium tuberculosis (M.tuberculosis) lineage 7, the differentially abundant proteins in strains of M. tuberculosis lineage 7 and lineage 4 were defined. Comparative proteomic analysis by mass spectrometry was employed to identify, quantitate and compare the protein profiles of strains from the two M. tuberculosis lineages. Label-free peptide quantification of whole cells from M. tuberculosis lineage 7 and 4 yielded the identification of 2825 and 2541 proteins, respectively. A combined total of 2867 protein groups covering 71% of the predicted M. tuberculosis proteome were identified. The abundance of 125 proteins in M. tuberculosis lineage 7 and 4 strains was significantly altered. Notably, the analysis showed that a number of M. tuberculosis proteins involved in growth and virulence were less abundant in lineage 7 strains compared to lineage 4. Five ABC transporter proteins, three phosphate binding proteins essential for inorganic phosphate uptake, and six components of the type 7 secretion system ESX-3 involved in iron acquisition were less abundant in M. tuberculosis lineage 7. This proteogenomic analysis provided an insight into the lineage 7-specific protein profile which may provide clues to understanding the differential properties of lineage 7 strains in terms of slow growth, survival fitness, and pathogenesis.

Comparative proteomic analysis of Neisseria meningitidis wildtype and dprA null mutant strains links DNA processing to pilus biogenesis.

BACKGROUND: DNA processing chain A (DprA) is a DNA binding protein which is ubiquitous in bacteria, and is required for DNA transformation to various extents among bacterial species. However, the interaction of DprA with competence and recombination proteins is poorly understood. Therefore, the proteomes of whole Neisseria meningitidis (Nm) wildtype and dprA mutant cells were compared. Such a comparative proteomic analysis increases our understanding of the interactions of DprA with other Nm components and may elucidate its potential role beyond DNA processing in transformation. RESULTS: Using label-free quantitative proteomics, a total of 1057 unique Nm proteins were identified, out of which 100 were quantified as differentially abundant (P ≤ 0.05 and fold change ≥ |2|) in the dprA null mutant. Proteins involved in homologous recombination (RecA, UvrD and HolA), pilus biogenesis (PilG, PilT1, PilT2, PilM, PilO, PilQ, PilF and PilE), cell division, including core energy metabolism, and response to oxidative stress were downregulated in the Nm dprA null mutant. The mass spectrometry data are available via ProteomeXchange with identifier PXD006121. Immunoblotting and co-immunoprecipitation were employed to validate the association of DprA with PilG. The analysis revealed reduced amounts of PilG in the dprA null mutant and reduced amounts of DprA in the Nm pilG null mutant. Moreover, a number of pilus biogenesis proteins were shown to interact with DprA and /or PilG. CONCLUSIONS: DprA interacts with proteins essential for Nm DNA recombination in transformation, pilus biogenesis, and other functions associated with the inner membrane. Inverse downregulation of Nm DprA and PilG expression in the corresponding mutants indicates a link between DNA processing and pilus biogenesis.

Characterization of the Neisseria meningitidis Helicase RecG.

Neisseria meningitidis (Nm) is a Gram-negative oral commensal that opportunistically can cause septicaemia and/or meningitis. Here, we overexpressed, purified and characterized the Nm DNA repair/recombination helicase RecG (RecGNm) and examined its role during genotoxic stress. RecGNm possessed ATP-dependent DNA binding and unwinding activities in vitro on a variety of DNA model substrates including a Holliday junction (HJ). Database searching of the Nm genomes identified 49 single nucleotide polymorphisms (SNPs) in the recGNm including 37 non-synonymous SNPs (nsSNPs), and 7 of the nsSNPs were located in the codons for conserved active site residues of RecGNm. A transient reduction in transformation of DNA was observed in the Nm ΔrecG strain as compared to the wildtype. The gene encoding recGNm also contained an unusually high number of the DNA uptake sequence (DUS) that facilitate transformation in neisserial species. The differentially abundant protein profiles of the Nm wildtype and ΔrecG strains suggest that expression of RecGNm might be linked to expression of other proteins involved in DNA repair, recombination and replication, pilus biogenesis, glycan biosynthesis and ribosomal activity. This might explain the growth defect that was observed in the Nm ΔrecG null mutant.

Label-free based quantitative proteomics analysis of primary neonatal porcine Leydig cells exposed to the persistent contaminant 3-methylsulfonyl-DDE.

Evidence that persistent environmental pollutants may target the male reproductive system is increasing. The male reproductive system is regulated by secretion of testosterone by testicular Leydig cells, and perturbation of Leydig cell function may have ultimate consequences. 3-Methylsulfonyl-DDE (3-MeSO2-DDE) is a potent adrenal toxicants formed from the persistent insecticide DDT. Although studies have revealed the endocrine disruptive effect of 3-MeSO2-DDE, the underlying mechanisms at cellular level in steroidogenic Leydig cells remains to be established. The current study addresses the effect of 3-MeSO2-DDE on viability, hormone production and proteome response of primary neonatal porcine Leydig cells. The AlamarBlue™ assay was used to evaluate cell viability. Solid phase radioimmunoassay was used to measure concentration of hormones produced by both unstimulated and Luteinizing hormone (LH)-stimulated Leydig cells following 48h exposure. Protein samples from Leydig cells exposed to a non-cytotoxic concentration of 3-MeSO2-DDE (10 µM) were subjected to nano-LC-MS/MS and analyzed on a Q Exactive mass spectrometer and quantified using label-free quantitative algorithm. Gene Ontology (GO) and Ingenuity Pathway Analysis (IPA) were carried out for functional annotation and identification of protein interaction networks. 3-MeSO2-DDE regulated Leydig cell steroidogenesis differentially depending on cell culture condition. Whereas its effect on testosterone secretion at basal condition was stimulatory, the effect on LH-stimulated cells was inhibitory. From triplicate experiments, a total of 6804 proteins were identified in which the abundance of 86 proteins in unstimulated Leydig cells and 145 proteins in LH-stimulated Leydig cells was found to be significantly regulated in response to 3-MeSO2-DDE exposure. These proteins not only are the first reported in relation to 3-MeSO2-DDE exposure, but also display small number of proteins shared between culture conditions, suggesting the action of 3-MeSO2-DDE on several targeted pathways, including mitochondrial dysfunction, oxidative phosphorylation, EIF2-signaling, and glutathione-mediated detoxification. Further identification and characterization of these proteins and pathways may build our understanding to the molecular basis of 3-MeSO2-DDE induced endocrine disruption in Leydig cells.

An investigation of the endocrine disrupting potential of enniatin B using in vitro bioassays.

Evidence that some of the fungal metabolites present in food and feed may act as potential endocrine disruptors is increasing. Enniatin B (ENN B) is among the emerging Fusarium mycotoxins known to contaminate cereals. In this study, the H295R and neonatal porcine Leydig cell (LC) models, and reporter gene assays (RGAs) have been used to investigate the endocrine disrupting activity of ENN B. Aspects of cell viability, cell cycle distribution, hormone production as well as the expression of key steroidogenic genes were assessed using the H295R cell model. Cell viability and hormone production levels were determined in the LC model, while cell viability and steroid hormone nuclear receptor transcriptional activity were measured using the RGAs. ENN B (0.01-100µM) was cytotoxic in the H295R and LC models used; following 48h incubation with 100µM. Flow cytometry analysis showed that ENN B exposure (0.1-25µM) led to an increased proportion of cells in the S phase at higher ENN B doses (>10µM) while cells at G0/G1 phase were reduced. At the receptor level, ENN B (0.00156-15.6µM) did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs), however cell viability was affected at 15.6µM. Measurement of hormone levels in H295R cells revealed that the production of progesterone, testosterone and cortisol in exposed cells were reduced, but the level of estradiol was not significantly affected. There was a general reduction of estradiol and testosterone levels in exposed LC. Only the highest dose (100µM) used had a significant effect, suggesting the observed inhibitory effect is more likely associated with the cytotoxic effect observed at this dose. Gene transcription analysis in H295R cells showed that twelve of the sixteen genes were significantly modulated (p<0.05) by ENN B (10µM) compared to the control. Genes HMGR, StAR, CYP11A, 3ßHSD2 and CYP17 were downregulated, whereas the expression of CYP1A1, NR0B1, MC2R, CYP21, CYP11B1, CYP11B2 and CYP19 were upregulated. The reduction of hormones and modulation of genes at the lower dose (10µM) in the H295R cells suggests that adrenal endocrine toxicity is an important potential hazard.

Using SILAC proteomics to investigate the effect of the mycotoxin, alternariol, in the human H295R steroidogenesis model.

The mycotoxin alternariol (AOH) is an important contaminant of fruits and cereal products. The current study sought to address the effect of a non-toxic AOH concentration on the proteome of the steroidogenic H295R cell model. Quantitative proteomics based on stable isotope labeling by amino acids in cell culture (SILAC) coupled to 1D-SDS-PAGE-LC-MS/MS was applied to subcellular-enriched protein samples. Gene ontology (GO) and ingenuity pathway analysis (IPA) were further carried out for functional annotation and identification of protein interaction networks. Furthermore, the effect of AOH on apoptosis and cell cycle distribution was also determined by the use of flow cytometry analysis. This work identified 22 proteins that were regulated significantly. The regulated proteins are those involved in early stages of steroid biosynthesis (SOAT1, NPC1, and ACBD5) and C21-steroid hormone metabolism (CYP21A2 and HSD3B1). In addition, several proteins known to play a role in cellular assembly, organization, protein synthesis, and cell cycle were regulated. These findings provide a new framework for studying the mechanisms by which AOH modulates steroidogenesis in H295R cell model.