BIOF-HILO Assay: A New MALDI-TOF Mass Spectrometry Based Method for Discriminating Between High- and Low-Biofilm-Producing Candida parapsilosis Isolates.

Candida parapsilosis is the most frequent cause of catheter-related candidemia among non-Candida albicans species. This may be related to intrinsic capabilities as adhering and forming a biofilm on abiotic surfaces such as on medical devices. As previously demonstrated, patients infected with high biofilm-producing C. parapsilosis isolates had a greater mortality risk compared to patients infected with low biofilm-producing C. parapsilosis isolates. We developed the BIOF-HILO assay, a MALDI-TOF mass spectrometry (MS)-based assay, which compares mass spectra obtained from attached and suspended isolate cells during the early (i.e., 3-h) adhesion phase of in vitro biofilm formation. The composite correlation index (CCI) analysis was used to discriminate between mass spectra differences of the two cell types, classifying all 50 C. parapsilosis clinical isolates, included in the study, after only 3-h of testing, in high or low biofilm producers. All high (n = 25) or low (n = 25) biofilm producers had, according to CCI mass spectra comparison values, higher or lower than one CCI ratios, which were obtained by dividing the CCIsuspended cells by the CCIattached cells. In conclusion, the BIOF-HILO assay allows a rapid categorization of C. parapsilosis clinical isolates in high or low biofilm producers. This information, if timely provided to physicians, may improve treatment outcomes in patients with C. parapsilosis candidemia.

Entamoeba dispar: A Rare Case of Enteritis in a Patient Living in a Nonendemic Area.

Entamoeba dispar, a common noninvasive parasite, is indistinguishable in its cysts and trophozoite forms from Entamoeba histolytica, the cause of invasive amebiasis, by microscopy. To differentiate the two species seems to be a problem for laboratory diagnosis. Recent experimental studies showed that E. dispar can be considered pathogenic too. We present a rare case of enteritis due to E. dispar.

PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis.

The role and function of PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) remains elusive. In this study for the first time, Mtb isogenic mutants missing selected PE_PGRSs were used to investigate their role in the pathogenesis of tuberculosis (TB). We demonstrate that the MtbΔPE_PGRS30 mutant was impaired in its ability to colonize lung tissue and to cause tissue damage, specifically during the chronic steps of infection. Inactivation of PE_PGRS30 resulted in an attenuated phenotype in murine and human macrophages due to the inability of the Mtb mutant to inhibit phagosome-lysosome fusion. Using a series of functional deletion mutants of PE_PGRS30 to complement MtbΔPE_PGRS30, we show that the unique C-terminal domain of the protein is not required for the full virulence. Interestingly, when Mycobacterium smegmatis recombinant strain expressing PE_PGRS30 was used to infect macrophages or mice in vivo, we observed enhanced cytotoxicity and cell death, and this effect was dependent upon the PGRS domain of the protein.Taken together these results indicate that PE_PGRS30 is necessary for the full virulence of Mtb and sufficient to induce cell death in host cells by the otherwise non-pathogenic species M. smegmatis, clearly demonstrating that PE_PGRS30 is an Mtb virulence factor.

PPE_MPTR genes are differentially expressed by Mycobacterium tuberculosis in vivo.

The PPE_MPTR protein sub-family is unique to mycobacteria and comprises proteins found only in MTB complex and in few other pathogenic mycobacteria. Very little is known about the precise function of PPE_MPTR, as well as on the expression pattern and the transcriptional regulation of their structural genes. In the present work, real time RT-PCR techniques were used to determine the expression profile of PPE_MPTR genes of Mycobacterium tuberculosis during infection in vivo and in different culture conditions. The PPE_MPTR genes showed a similar expression profile in axenic cultures, with a significant increase of gene expression following exposure to environmental signals such as SDS, isoniazid and ethambutol. The PPE_MPTR genes were expressed in lung and spleen tissues infected by M. tuberculosis, and levels of expression were similar to those of genes encoding M. tuberculosis virulence factors such as hbhA and mpt64. Levels and pattern of gene expression in host tissues were different for each PPE_MPTR gene under study. The results of this study indicate that PPE_MPTR genes are differentially regulated in the lung and spleen tissues during M. tuberculosis infection, suggesting that each gene responds independently to the different and complex environmental signals encountered in host tissues.

Structure and functional regulation of RipA, a mycobacterial enzyme essential for daughter cell separation.

Cell separation depends on cell-wall hydrolases that cleave the peptidoglycan layer connecting daughter cells. In Mycobacterium tuberculosis, this process is governed by the predicted endopeptidase RipA. In the absence of this enzyme, the bacterium is unable to divide and exhibits an abnormal phenotype. We here report the crystal structure of a relevant portion of RipA, containing its catalytic-domain and an extra-domain of hitherto unknown function. The structure clearly demonstrates that RipA is produced as a zymogen, which needs to be activated to achieve cell-division. Bacterial cell-wall degradation assays and proteolysis experiments strongly suggest that activation occurs via proteolytic processing of a fully solvent exposed loop identified in the crystal structure. Indeed, proteolytic cleavage at this loop produces an activated form, consisting of the sole catalytic domain. Our work provides the first evidence of self-inhibition in cell-disconnecting enzymes and opens a field for the design of novel antitubercular therapeutics.