A shift in the virulence potential of Corynebacterium pseudotuberculosis biovar ovis after passage in a murine host demonstrated through comparative proteomics.

BACKGROUND: Corynebacterium pseudotuberculosis biovar ovis, a facultative intracellular pathogen, is the etiologic agent of caseous lymphadenitis in small ruminants. During the infection process, C. pseudotuberculosis changes its gene expression to resist different types of stresses and to evade the immune system of the host. However, factors contributing to the infectious process of this pathogen are still poorly documented. To better understand the C. pseudotuberculosis infection process and to identify potential factors which could be involved in its virulence, experimental infection was carried out in a murine model using the strain 1002_ovis and followed by a comparative proteomic analysis of the strain before and after passage. RESULTS: The experimental infection assays revealed that strain 1002_ovis exhibits low virulence potential. However, the strain recovered from the spleen of infected mice and used in a new infection challenge showed a dramatic change in its virulence potential. Label-free proteomic analysis of the culture supernatants of strain 1002_ovis before and after passage in mice revealed that 118 proteins were differentially expressed. The proteome exclusive to the recovered strain contained important virulence factors such as CP40 proteinase and phospholipase D exotoxin, the major virulence factor of C. pseudotuberculosis. Also, the proteome from recovered condition revealed different classes of proteins involved in detoxification processes, pathogenesis and export pathways, indicating the presence of distinct mechanisms that could contribute in the infectious process of this pathogen. CONCLUSIONS: This study shows that C. pseudotuberculosis modifies its proteomic profile in the laboratory versus infection conditions and adapts to the host context during the infection process. The screening proteomic performed us enable identify known virulence factors, as well as potential proteins that could be related to virulence this pathogen. These results enhance our understanding of the factors that might influence in the virulence of C. pseudotuberculosis.

Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data.

Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched for identification of the most stably expressed genes of a given bacterium. Herein, we searched a RNA-seq dataset from various experiments with the pathogenic bacterium Corynebacterium pseudotuberculosis, grown under different stress conditions, in order to select appropriate candidate reference genes for this species. Nineteen genes involved in maintenance of basic cellular functions, so-called housekeeping genes, were chosen for study and their expression profiles in C. pseudotuberculosis were evaluated throughout all growth conditions. Eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, matched the defined criteria to be included as candidate reference genes. Transcriptional levels of these genes were quantified by RT-qPCR assays after growth of C. pseudotuberculosis under two additional conditions. Expression stability analysis by NormFinder indicated the combination of genes encoding DNA gyrase subunit A (gyrA) and elongation factor P (fusA) as the most suitable for normalization of RT-qPCR studies in C. pseudotuberculosis.

Diagnosis of bacteria from the CMNR group in farm animals.

The CMNR group comprises bacteria of the genera Corynebacterium, Mycobacterium, Nocardia, and Rhodococcus and share cell wall and DNA content characteristics. Many pathogenic CMNR bacteria cause diseases such as mastitis, lymphadenitis, and pneumonia in farmed animals, which cause economic losses for breeders and represent a threat to public health. Traditional diagnosis in CMNR involves isolating target bacteria on general or selective media and conducting metabolic analyses with the assistance of laboratory biochemical identification systems. Advanced mass spectrometry may also support diagnosing these bacteria in the clinic's daily routine despite some challenges, such as the need for isolated bacteria. In difficult identification among some CMNR members, molecular methods using polymerase chain reaction (PCR) emerge as reliable options for correct specification that is sometimes achieved directly from clinical samples such as tracheobronchial aspirates and feces. On the other hand, immunological diagnostics such as the skin test or Enzyme-Linked Immunosorbent Assay (ELISA) for Mycobacterium tuberculosis yield promising results in subclinical infections with no bacterial growth involved. In this review, we present the methods most commonly used to diagnose pathogenic CMNR bacteria and discuss their advantages and limitations, as well as challenges and perspectives on adopting new technologies in diagnostics.

Identification of 11 new exoproteins in Corynebacterium pseudotuberculosis by comparative analysis of the exoproteome.

This study involves the comparison between the exoproteomes of two different strains of Corynebacterium pseudotuberculosis, the etiologic agent of caseous lymphadenitis in small ruminants. In a previous study, based on a gel-free system (TPP-LC/MS(E)), 70 exoproteins for the strain 1002 and 67 for the strain C231, totaling 93 different extracellular proteins for C. pseudotuberculosis, were identified. In the present work, we have used 2D gel electrophoresis to resolve the extracellular proteins of both strains, which were then digested with trypsin, analyzed by MALDI-TOF/TOF and identified with the software MASCOT(®). A total of 45 extracellular proteins of C. pseudotuberculosis were identified by this approach. The comparative analysis between the strains 1002 and C231 identified 13 and 3 strain-specific proteins, respectively, 11 of which are novel. These newly identified proteins may play an important role in the physiology and virulence of C. pseudotuberculosis.

Differential Exoproteome analysis of two corynebacterium pseudotuberculosis biovar ovis strains isolated from goat (1002) and sheep (C231).

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis a chronic infectious disease affecting small ruminants. The 2D-DIGE technique was used to compare the exoproteomes of two C. pseudotuberculosis biovar ovis strains isolated from goat (strain 1002) and sheep (strain C231). Seventeen proteins differentially produced were identified here. Nine proteins appeared over-produced in the exoproteome of 1002 goat strain and 8 in that of C231 sheep strain. These proteins were related to various biological functions, such as the cell envelope, respiratory metabolism and proteolysis. This proteomic analysis revealed strain-specific exoproteins although each of the corresponding genes was found in both strain genomes. Such differential expression pattern may reflect inter-strain differences in adaptation to a specific host, in pathogenicity and or in antigenicity of this pathogenic bacterium.

Exploring the MALDI Biotyper for the Identification of Corynebacterium pseudotuberculosis biovar Ovis and Equi.

Biochemical, serological, and molecular methods have been developed for the laboratory diagnosis of diseases caused by C. pseudotuberculosis (CP), but the identification of the pathogen and biovars differentiation may be time-consuming, expensive, and confusing compared with other bacteria. This study aimed to evaluate MALDI Biotyper and Overall Genome Relatedness Index (OGRI) analysis to optimize the identification and differentiation of biovars of C. pseudotuberculosis. Out of 230 strains isolated from several hosts and countries, 202 (87.8%) were precisely classified using MALDI Biotyper and the BioNumerics platform. The classification accuracies for the Ovis and Equi biovars were 80 (88.75%) and 82 (92.68%), respectively. When analyzing a sampling of these strains by Average Nucleotide Identity based on BLAST and TETRA analyses using genomic sequence data, it was possible to differentiate 100% of the strains in Equi and Ovis. Our data show that MALDI Biotyper and OGRI analysis help identify C. pseudotuberculosis at the species and biovar levels.

A combined approach for comparative exoproteome analysis of Corynebacterium pseudotuberculosis.

BACKGROUND: Bacterial exported proteins represent key components of the host-pathogen interplay. Hence, we sought to implement a combined approach for characterizing the entire exoproteome of the pathogenic bacterium Corynebacterium pseudotuberculosis, the etiological agent of caseous lymphadenitis (CLA) in sheep and goats. RESULTS: An optimized protocol of three-phase partitioning (TPP) was used to obtain the C. pseudotuberculosis exoproteins, and a newly introduced method of data-independent MS acquisition (LC-MSE) was employed for protein identification and label-free quantification. Additionally, the recently developed tool SurfG+ was used for in silico prediction of sub-cellular localization of the identified proteins. In total, 93 different extracellular proteins of C. pseudotuberculosis were identified with high confidence by this strategy; 44 proteins were commonly identified in two different strains, isolated from distinct hosts, then composing a core C. pseudotuberculosis exoproteome. Analysis with the SurfG+ tool showed that more than 75% (70/93) of the identified proteins could be predicted as containing signals for active exportation. Moreover, evidence could be found for probable non-classical export of most of the remaining proteins. CONCLUSIONS: Comparative analyses of the exoproteomes of two C. pseudotuberculosis strains, in addition to comparison with other experimentally determined corynebacterial exoproteomes, were helpful to gain novel insights into the contribution of the exported proteins in the virulence of this bacterium. The results presented here compose the most comprehensive coverage of the exoproteome of a corynebacterial species so far.

Characterization of a new multidrug-resistant Brazilian K. pneumoniae isolate and 172 Klebsiella spp. sequenced strains: Genomic island, multilocus sequence typing and capsule locus dataset.

The genus Klebsiella comprises species that cause nosocomial and community-acquired infections. A dataset was created to compile the sequence type (ST) and capsule type (K-locus) information predicted for 172 worldwide isolates of Klebsiella spp. whose complete genomes could be retrieved from the GenBank (NCBI) repository. The dataset also includes information related to one multidrug-resistant strain (B31) isolated from a patient who was admitted to an intensive care unit in the Northeast region of Brazil. This strain was phenotypically characterized and submitted to whole-genome sequencing and comparative genomics analysis as we recently reported [1]. The dataset also compiles information on Pathogenicity Islands (PIs), Resistance Islands (RIs) and Miscellaneous Islands (MIS) present in the genome of strain B31. The information provided here may support outbreak prevention policies and future epidemiological studies involving Klebsiella spp.

Comparative genomics with a multidrug-resistant Klebsiella pneumoniae isolate reveals the panorama of unexplored diversity in Northeast Brazil.

The emergence of community acquired infections increases the public health concern on K. pneumoniae and closely related bacteria among which antimicrobial resistance spreads. We report a multidrug-resistant K. pneumoniae isolate, B31, of a patient infected in the community and admitted to an intensive care unit in Northeast Brazil. Antimicrobial susceptibility and genome information were thoroughly investigated to characterize B31 in front of 172 sequenced strains of different countries. Assigned to the Sequence Type 15, which is globally spread, B31 presented extended spectrum beta-lactamase, tigecycline and ciprofloxacin resistance. Genome sequencing revealed most resistance genes being carried by plasmids with high dissemination potential. The absence of main virulence factors, like yersiniabactin and colibactin, apparently suggests a mild pathogenic strain which, on the contrary, persisted and caused severe infection in a previously healthy patient. The present study contributes to unveil the unclear genomic scenario of virulent and multidrug-resistant K. pneumoniae in Brazil.

Gene Expression Analysis in Bacteria by RT-qPCR.

Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) using fluorescent DNA-binding dyes is now a gold-standard methodology to study bacterial gene expression through relative quantitation of target mRNAs under specific experimental conditions, and recent developments in the technology allow for gene expression analysis in single cells. Nevertheless, several critical steps of the RT-qPCR protocol need to be carefully addressed in order to obtain reliable results, particularly regarding RNA sample quality and appropriate choice of reference genes. Besides, accurate reporting of study conditions is essential, as recommended by the MIQE guidelines. Herein, we provide a practical approach to quantitation of the transcript levels of bacterial genes using RT-qPCR, including a general protocol for obtaining good-quality bacterial RNA and a discussion on the selection and validation of candidate bacterial reference genes for data normalization.

Oral immunization with Salmonella harboring a Sm14-based DNA vaccine does not protect mice against Schistosoma mansoni infection.

The protection against Schistosoma mansoni infection was evaluated in SWISS mice orally vaccinated with an attenuated strain of Salmonella carrying a Sm14-based DNA vaccine. Although this formulation was not able to afford a reduction in the worm burden, a non-antigen-specific decrease in schistosome-induced granulomatous reaction was verified in livers of mice that received Salmonella.

NMR insights on nano silver post-surgical treatment of superficial caseous lymphadenitis in small ruminants.

Caseous lymphadenitis (CL), caused by a pathogen of the second class of biosafety - Corynebacterium pseudotuberculosis, is a chronic and severe infectious disease that affects small ruminants and requires long, ineffective treatment which generally leads to animal sacrifice so as to stop the disease spreading. The infected animals suffer the excision of affected superficial lymph nodes and post-surgical treatment with iodine (10% solution in ethanol) and, sometimes, prolonged antibiotic use, but only if the sick animals are of great importance to breeding. Herein, we propose a cheap and easy to apply treatment of CL with excellent results using biogenic silver nanoparticles (AgNP) based technology. AgNP antibacterial properties were investigated in vitro against Corynebacterium pseudotuberculosis cells and in vivo on small ruminants with CL. Treatment of surgical wounds resulting from the excision of superficial CL lesions with a AgNP-based cream was compared to the standard post-surgical treatment method by iodine. Also, the effects of AgNP-based cream treatment were evaluated and compared with the effects of the iodine CL treatment by serum NMR-based metabolomics. Serum samples were collected from 29 animals, 9 sheep and 20 goats, during the treatments and analyzed. All animals showed stable serum metabolomes when iodine or AgNP-based cream effects were compared. The AgNP-based cream treatment showed excellent results, especially in accelerating the healing of wounds, which occurred two to three times faster in comparison with the iodine treatment. AgNP-based cream treatment also prevented CL reappearance and did not cause any side effects on animals. This is the first report on very effective post-surgical treatment of superficial CL in small ruminants based on biogenic silver nanoparticles, which might open up the possibility for a safe veterinary application of AgNP-based cream.

Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples.

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 10(3) c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.

Two-Component Signal Transduction Systems of Pathogenic Bacteria As Targets for Antimicrobial Therapy: An Overview.

The bacterial communities in a wide range of environmental niches sense and respond to numerous external stimuli for their survival. Primarily, a source they require to follow up this communication is the two-component signal transduction system (TCS), which typically comprises a sensor Histidine kinase for receiving external input signals and a response regulator that conveys a proper change in the bacterial cell physiology. For numerous reasons, TCSs have ascended as convincing targets for antibacterial drug design. Several studies have shown that TCSs are essential for the coordinated expression of virulence factors and, in some cases, for bacterial viability and growth. It has also been reported that the expression of antibiotic resistance determinants may be regulated by some TCSs. In addition, as a mode of signal transduction, phosphorylation of histidine in bacteria differs from normal serine/threonine and tyrosine phosphorylation in higher eukaryotes. Several studies have shown the molecular mechanisms by which TCSs regulate virulence and antibiotic resistance in pathogenic bacteria. In this review, we list some of the characteristics of the bacterial TCSs and their involvement in virulence and antibiotic resistance. Furthermore, this review lists and discusses inhibitors that have been reported to target TCSs in pathogenic bacteria.

Characterization of the Opp peptide transporter of Corynebacterium pseudotuberculosis and its role in virulence and pathogenicity.

Despite the economic importance of caseous lymphadenitis (CLA), a chronic disease caused by Corynebacterium pseudotuberculosis, few genes related to the virulence of its etiologic agent have been characterized. The oligopeptide permease (Opp) transporters are located in the plasma membrane and have functions generally related to the uptake of peptides from the extracellular environment. These peptide transporters, in addition to having an important role in cell nutrition, also participate in the regulation of various processes involving intercellular signaling, including the control of the expression of virulence genes in pathogenic bacteria. To study the role of Opp in C. pseudotuberculosis, an OppD deficient strain was constructed via simple crossover with a nonreplicative plasmid carrying part of the oppD gene sequence. As occurred to the wild-type, the ΔoppD strain showed impaired growth when exposed to the toxic glutathione peptide (GSH), indicating two possible scenarios: (i) that this component can be internalized by the bacterium through an Opp-independent pathway or (ii) that there is toxicity while the peptide is extracellular. Additionally, the ΔoppD mutant presented a reduced ability to adhere to and infect macrophages compared to the wild-type, although both strains exhibit the same potential to colonize spleens and cause injury and death to infected mice.

Ion Torrent-based transcriptional assessment of a Corynebacterium pseudotuberculosis equi strain reveals denaturing high-performance liquid chromatography a promising rRNA depletion method.

Corynebacterium pseudotuberculosis equi is a Gram-positive pathogenic bacterium which affects a variety of hosts. Besides the great economic losses it causes to horse-breeders, this organism is also known to be an important infectious agent to cattle and buffaloes. As an outcome of the efforts in characterizing the molecular basis of its virulence, several complete genome sequences were made available in recent years, enabling the large-scale assessment of genes throughout distinct isolates. Meanwhile, the RNA-seq stood out as the technology of choice for comprehensive transcriptome studies, which may bring valuable information regarding active genomic regions, despite of the still impeditive associated costs. In an attempt to increase the use of generated reads per instrument run, by effectively eliminating unwanted rRNAs from total RNA samples without relying on any commercially available kits, we applied denaturing high-performance liquid chromatography (DHPLC) as an alternative method to assess the transcriptional profile of C. pseudotuberculosis. We have found that the DHPLC depletion method, allied to Ion Torrent sequencing, allows mapping of transcripts in a comprehensive way and identifying novel transcripts when a de novo approach is used. These data encourage us to use DHPLC in future transcriptional evaluations in C. pseudotuberculosis.

Progression of 'OMICS' methodologies for understanding the pathogenicity of Corynebacterium pseudotuberculosis: the Brazilian experience.

Since the first successful attempt at sequencing the Corynebacterium pseudotuberculosis genome, large amounts of genomic, transcriptomic and proteomic data have been generated. C. pseudotuberculosis is an interesting bacterium due to its great zoonotic potential and because it causes considerable economic losses worldwide. Furthermore, different strains of C. pseudotuberculosis are capable of causing various diseases in different hosts. Currently, we seek information about the phylogenetic relationships between different strains of C. pseudotuberculosis isolates from different hosts across the world and to employ these data to develop tools to diagnose and eradicate the diseases these strains cause. In this review, we present the latest findings on C. pseudotuberculosis that have been obtained with the most advanced techniques for sequencing and genomic organization. We also discuss the development of in silico tools for processing these data to prompt a better understanding of this pathogen.

A Role for Sigma Factor σ(E) in Corynebacterium pseudotuberculosis Resistance to Nitric Oxide/Peroxide Stress.

Pathogenic intracellular bacteria can respond to antimicrobial mechanisms of the host cell through transient activation of stress-responsive genes by alternative sigma (σ) factors of the RNA polymerase. We evaluated the contribution of the extracytoplasmic function sigma factor σ(E) for Corynebacterium pseudotuberculosis resistance to stress conditions resembling those found intracellularly during infection. A sigE-null mutant strain (ΔsigE) of this bacterium was more susceptible in vitro to acidic pH, cell surface stressors, and biologically relevant concentrations of nitric oxide (NO). The same mutant strain was unable to persist in C57BL/6 mice but remained infective in mice lacking inducible nitric oxide synthase (iNOS), confirming the significance of σ(E) for resistance to nitric oxide/peroxide stress in vivo. High-throughput proteomic analysis identified NO-responsive extracellular proteins of C. pseudotuberculosis and demonstrated the participation of σ(E) in composition of this bacterium's exoproteome.

Evidence for reductive genome evolution and lateral acquisition of virulence functions in two Corynebacterium pseudotuberculosis strains.

BACKGROUND: Corynebacterium pseudotuberculosis, a gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. METHODOLOGY AND FINDINGS: We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. CONCLUSIONS: These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.