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Nematophagous fungi are commonly used as biological control agents of plant and animal parasitic nematodes. However, relatively little is known of the environmental attributes conferring pathogenicity in these fungi. In this report, we investigated the role of PacC-mediated pH response in the pathogenesis of the nematophagous fungus Clonostachys rosea. We identified a pacC orthologue from this fungus and found that its transcript was elevated in C. rosea during the early stage of its infection of nematode. Disruption of pacC resulted in slowed growth at alkaline pH, altered filamentation, reduced conidiation and attenuated virulence to nematodes. The expression of an extracellular serine protease PrC, a putative virulence factor, was downregulated in the pacC mutants. The PrC transcript levels were significantly higher under alkaline growth conditions than under acidic growth conditions. Promoter activity analysis and electrophoretic mobility shift assay indicated that the regulation of PrC by pH via the PacC pathway occurred at the transcriptional level. In conclusion, PacC functions as a positive regulator of virulence to nematodes in C. rosea.

Nematophagous fungi have been used as biological control agents against nematodes parasitic to plants and animals. These fungi can secret subtilisin-like extracellular serine proteases during the infection of nematodes. The expression of these subtilisin-like serine proteases is regulated by nitrogen sources, including nematode cuticle. However, the mechanisms underlying the nitrogen sources-induced expression of these serine proteases is not well understood. In this study, we investigated the effect of nitrogen sources on the expression of a subtilisin-like extracellular protease, prC, in the nematophagous fungus Clonostachys rosea. Disruption of prC attenuated infection of the fungus to nematodes, indicating that this gene functions as a virulence factor. The inhibition of basal expression of prC by the preferred nitrogen sources (glutamine, ammonia) occurred at the transcriptional level. In contrast, nematode cuticle induced the expression of prC at the post-transcriptional level. The inducible expression of prC by nematode cuticle was significantly suppressed by glutamine, ammonia and phenylmethylsulfonyl fluoride (an inhibitor of serine protease). Thus, the existence of active PrC, albeit at a low level in the medium, is probably essential for further induction of this gene by nematode cuticle. Moreover, the low molecule weight (< 3 kD) degradation products of nematode cuticle could significantly induce the expression of prC. Ammonia suppresses the virulence of C. rosea against nematodes, probably by inhibiting prC expression. Thus, the nematophagous fungi probably could not function well as biocontrol agents in fields fertilized with a large amount of ammonium salt.

The aim of this study was to discover novel biomarkers for pulmonary tuberculosis (TB). Differentially expressed proteins in the serum of patients with TB were screened and identified by iTRAQ-two dimensional liquid chromatography tandem mass spectrometry analysis. A total of 79 abnormal proteins were discovered in patients with TB compared with healthy controls. Of these, significant differences were observed in 47 abnormally expressed proteins between patients with TB or pneumonia and chronic obstructive pulmonary disease (COPD). Patients with TB (n = 136) exhibited significantly higher levels of serum amyloid A (SAA), vitamin K-dependent protein Z (PROZ), and C4b-binding protein β chain (C4BPB) than those in healthy controls (n = 66) (P<0.0001 for each) albeit significantly lower levels compared with those in patients with pneumonia (n = 72) (P<0.0001 for each) or COPD (n = 72) (P<0.0001, P<0.0001, P = 0.0016, respectively). After 6 months of treatment, the levels of SAA and PROZ were significantly increased (P = 0.022, P<0.0001, respectively), whereas the level of C4BPB was significantly decreased (P = 0.0038) in treated TB cases (n = 72). Clinical analysis showed that there were significant differences in blood clotting and lipid indices in patients with TB compared with healthy controls, patients with pneumonia or COPD, and treated TB cases (P<0.05). Correlation analysis revealed significant correlations between PROZ and INR (rs = 0.414, P = 0.044), and between C4BPB and FIB (rs = 0.617, P = 0.0002) in patients with TB. Receiver operating characteristic curve analysis revealed that the area under the curve value of the diagnostic model combining SAA, PROZ, and C4BPB to discriminate the TB group from the healthy control, pneumonia, COPD, and cured TB groups was 0.972, 0.928, 0.957, and 0.969, respectively. Together, these results suggested that SAA, PROZ, and C4BPB may serve as new potential biomarkers for TB. Our study may thus provide experimental data for the differential diagnosis of TB.

Pulmonary tuberculosis (TB) is among the diseases with the highest morbidity and mortality worldwide. Effective diagnostic methods for TB are lacking. In this study, we investigated long non-coding RNAs (lncRNAs) in plasma using microarray and the potential diagnostic value of lncRNAs for TB. We found a total of 163 up-regulated lncRNAs and 348 down-regulated lncRNAs. Gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and coding-noncoding co-expression (CNC) analyses showed that functions of differentially expressed lncRNAs were mainly enriched in the regulation of alpha-beta T cell activation and the T cell receptor signalling pathway. Four differentially expressed lncRNAs, NR_038221 (fold change = 3.79, P < 0.01), NR_003142 (fold change = 1.69, P < 0.05), ENST00000570366 (fold change = 3.04, P < 0.05), and ENST00000422183 (fold change = 2.11, P < 0.001), were verified using RT-qPCR. Among those, NR_038221, NR_003142, and ENST00000570366 were found to be up-regulated, while ENST00000422183 was down-regulated. The value of the area under the curve (AUC) for the diagnostic model consisting of the four lncRNAs was 0.845 (sensitivity = 79.2%, specificity = 75%). We further predicted 85 mRNAs and 404 miRNAs that potentially interact with these lncRNAs. Our study revealed the potential value of lncRNAs as biomarkers for early diagnosis of TB and the underlying mechanisms of these abnormally expressed lncRNAs in the pathogenesis of TB.

Rapid diagnosis of pulmonary tuberculosis is an effective measure to prevent the spread of tuberculosis. However, the grim fact is that the new, rapid, and safe methods for clinical diagnosis are lacking. Moreover, although auto-lysosome is critical in clearing Mycobacterium tuberculosis , the pathological significance of microRNAs, as biomarkers of tuberculosis, in autophagosome maturation is unclear. Here, these microRNAs were investigated by Solexa sequencing and qPCR validation, and a potential diagnostic model was established by logistic regression. Besides that, the mechanism of one of the microRNAs involved in the occurrence of tuberculosis was studied. The results showed that the expression of miR-423-5p, miR-17-5p, and miR-20b-5p were significantly increased in the serum of patients with tuberculosis. The combination of these three microRNAs established a model to diagnose tuberculosis with an accuracy of 78.18%, and an area under the curve value of 0.908. Bioinformatics analysis unveiled miR-423-5p as the most likely candidate in regulating autophagosome maturation. The up-regulation of miR-423-5p could inhibit autophagosome maturation through suppressing autophagosome–lysosome fusion in macrophages. Further investigations showed that VPS33A was the direct target of miR-423-5p, and the two CUGCCCCUC domains in VPS33A 3’-UTR were the direct regulatory sites for miR-423-5p. In addition, an inverse correlation between VPS33A and miR-423-5p was found in peripheral blood mononuclear cells of patients with tuberculosis. Since the inhibition of autolysosome formation plays a critical role in tuberculosis occurrence, our findings suggests that miR-423-5p could suppress autophagosome–lysosome fusion by post-transcriptional regulation of VPS33A , which might be important for the occurrence of active tuberculosis.

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