Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate.

Despite being the subject of intensive research, tuberculosis, caused by Mycobacterium tuberculosis, remains at present the leading cause of death from an infectious agent. Secreted and cell wall proteins interact with the host and play important roles in pathogenicity. These proteins are explored as candidate diagnostic markers, potential drug targets or vaccine antigens, and more recently special attention is being given to the role of their post-translational modifications. With the purpose of contributing to the proteomic and glycoproteomic characterization of this important pathogen, we performed a shotgun analysis of culture filtrate proteins of M. tuberculosis based on a liquid nano-HPLC tandem mass spectrometry and a label-free spectral counting normalization approach for protein quantification. We identified 1314 M. tuberculosis proteins in culture filtrate and found that the most abundant proteins belong to the extracellular region or cell wall compartment, and that the functional categories with higher protein abundance factor were virulence, detoxification and adaptation, and cell wall and cell processes. We could identify a group of proteins consistently detected in previous studies, most of which were highly abundant proteins. In culture filtrate, 140 proteins were predicted to contain one of the three types of bacterial N-terminal signal peptides. Besides, various proteins belonging to the ESX secretion systems, and to the PE and PPE families, secreted by the type VII secretion system using nonclassical secretion signals, were also identified. O-glycosylation was identified in 46 proteins, many of them lipoproteins and cell wall associated proteins. Finally, we provide proteomic evidence for 33 novel O-glycosylated proteins, aiding to the glycoproteomic characterization of relevant antigenic membrane and exported proteins. These findings are expected to collaborate with the research on pathogen derived biomarkers, virulence factors and vaccine candidates, and to provide clues to the understanding of the pathogenesis and survival strategies adopted by M. tuberculosis.

Identification of Leukotoxin and other vaccine candidate proteins in a Mannheimia haemolytica commercial antigen.

Bovine Respiratory Disease is the most costly disease that affects beef and dairy cattle industry. Its etiology is multifactorial, arising from predisposing environmental stress conditions as well as the action of several different respiratory pathogens. This situation has hindered the development of effective control strategies. Although different type of vaccines are available, many currently marketed vaccines are based on inactivated cultures of the main viral and bacterial agents involved in this pathology. The molecular composition of commercial veterinary vaccines is a critical issue. The present work aims to define at the proteomic level the most relevant valence of a line of commercial respiratory vaccines widely used in Central and South America. Since Mannheimia haemolytica is responsible for most of the disease associated morbid-mortality, we focused on the main proteins secreted by this pathogen, in particular Leukotoxin A, its main virulence factor. By Western blot analysis and mass spectrometry, Leukotoxin A was identified as a major component of M. haemolytica culture supernatants. We also identified other ten M. haemolytica proteins, including outer membrane proteins, periplasmic transmembrane solute transporters and iron binding proteins, which are relevant to achieve protective immunity against the pathogen. This work allowed a detailed molecular characterization of this vaccine component, providing evidence of its quality and efficacy. Furthermore, our results contributed to the identification of several proteins of interest as subunit vaccine candidates.

Pathogen-derived biomarkers for active tuberculosis diagnosis.

Tuberculosis (TB) is an infectious disease caused by members of Mycobacterium tuberculosis complex. Despite the availability of effective treatments, TB remains a major public health concern in most low and middle-income countries, representing worldwide the second leading cause of death from an infectious disease. Inadequate case detection and failures to classify the disease status hamper proper TB control. The limitations of the conventional diagnostic methods have encouraged much research activities in this field, but there is still an urgent need for an accurate point of care test for active TB diagnosis. A rapid, precise, and inexpensive TB diagnostic test would allow an earlier implementation of an appropriate treatment and the reduction of disease transmission. Pathogen-derived molecules present in clinical specimens of affected patients are being validated for that purpose. This short review aims to summarize the available data regarding biomarkers derived from M. tuberculosis, and their current usage in active TB diagnosis.

Association of the p53 codon 72 polymorphism with clinicopathological characteristics of colorectal cancer through mRNA analysis.

TP53 represents a suitable candidate for a colorectal cancer susceptibility locus. The polymorphism in the p53 72nd codon involves a proline to arginine substitution, leading to changes in gene transcription activity, interaction with other proteins and modulation of apoptosis. Studies evaluating the association between this polymorphism and colorectal cancer (CRC) have shown inconsistent results, and none have evaluated the mRNA status of TP53. The aim of the present study was to evaluate the association between this SNP expression at the mRNA level in CRC samples and patient clinicopathological variables and prognosis, p53 protein expression and TP53 mutation. This is the first report to describe the mRNA expression of p53 codon 72 alleles in CRC. We evaluated 101 non-related patients with CRC treated at the A.C. Camargo Cancer Center in Brazil. RNA was isolated from frozen tumor tissues using a trizol-based protocol. The polymorphism was detected using RT-PCR followed by Sanger sequencing. Associations were analyzed using Pearson's Chi-square or Fisher's exact tests, logistic regression and Cox. This polymorphism was significantly associated with clinicopathological variables related to increased tumor aggressiveness. The expression of Arg72 (OR, 3.83; CI 1.02-14.35; P=0.046) and the TNM stage (OR, 7.15; CI 1.45-35.29; P=0.016) were found to be independent predictors for recurrence. These data suggest that the mRNA expression of the Pro72 allele is associated with less favorable tumor features. The allele frequency of the p53 Pro72 was 0.26. The analysis of mRNA is important to determine the specific contribution of the allele expressed. These results suggest that this polymorphism may play a role in CRC.

Different mutation profiles associated to P53 accumulation in colorectal cancer.

The tumor suppressor TP53 gene is one of the most frequently mutated in different types of human cancer. Particularly in colorectal cancer (CRC), it is believed that TP53 mutations play a role in the adenoma-carcinoma transition of tumors during pathological process. In order to analyze TP53 expressed alleles in CRC, we examined TP53 mRNA in tumor samples from 101 patients with sporadic CRC. Samples were divided in two groups defined according to whether they exhibit positive or negative P53 protein expression as detected by immunohistochemistry (IHC). The presence of TP53 mutation was a common event in tumors with an overall frequency of 54.5%. By direct sequencing, we report 42 different TP53 sequence changes in 55 CRC patients, being two of them validated polymorphisms. TP53 mutations were more frequent in positive than in negative P53 detection group (p<0.0001), being the precise figures 79.6% and 30.8%, respectively. In addition, the mutation profiles were also different between the two groups of samples; while most of the mutations detected in P53 positive group were missense (38 out of 39), changes in P53 negative detection group include 7 insertions/deletions, 6 missense, 2 nonsense and 1 silent mutation. As previously observed, most mutations were concentrated in regions encoding P53 DNA binding domain (DBD). Codons 175, 248 and 273 together account for 36.7% of point mutations, in agreement with previous observations provided that these codons are considered mutation hotspots. Interestingly, we detected two new deletions and two new insertions. In addition, in three samples we detected two deletions and one insertion that could be explained as putative splicing variants or splicing errors.

Prenatal and postnatal characterization of a de novo Xq22.1 terminal deletion.

We present a case of a de novo Xq22.1 chromosomal terminal deletion discovered prenatally by conventional cytogenetics. The pregnancy resulted in the birth of a normal girl. Preferential inactivation of the abnormal X was demonstrated postnatally. Fluorescence in situ hybridization (FISH) demonstrated a terminal Xq deletion spanning Xq22.1 -->qter. An X painting probe ruled out a translocation. The deleted X chromosome was determined to be of paternal origin. The girl is now 4 years old with normal physical and psychomotor development. X chromosomal deletions are infrequent findings in prenatal diagnosis and present a difficult counseling challenge when they occur. Prenatal X-inactivation studies provide an opportunity for more informative genetic counseling when a de novo X chromosome deletion is detected.

Characterization of the immune response induced by a carbohydrate enriched fraction from Echinococcus granulosus protoscoleces in patients with cystic hydatid disease.

The serological and cellular immune response against a carbohydrate-enriched fraction of Echinococcus granulosus (E4+) was studied in a group of patients with cystic hydatid disease (CHD). The profile of IgG subclass against E4+ and the native non-enriched extract (protoscolex somatic antigen, PSA) were compared. A relatively higher ratio of IgG2:IgG4 was induced by E4+ than by PSA (12.4 vs 3.6 respectively). Serological data were associated with clinical parameters dealing with the outcome of disease. The expression of IgG1 and IgG4 subclasses against both of the antigens was associated with the progression of the disease. Interestingly, anti-PSA IgG4 was the only variable that was specifically associated with the stage of cysts (P < 0.04). Similar levels of IL-13 were evoked by peripheral blood mononuclear cells from patients with CHD when incubated with either of the antigens, while higher levels of IL-10 were obtained in supernatants from cells stimulated with E4+ (P < 0.029) than from those stimulated by PSA. Considerable amounts of IL-10 (median 60 pg/ml) were obtained in the supernatants of cells from healthy individuals incubated with E4+. Our results suggest a putative role for E4+ in immunoregulation during the course of infection with E. granulosus in humans.