Telomeres play important roles in genome stability and cell proliferation. Telomere lengths are heterogeneous and because just a few abnormal telomeres are sufficient to trigger significant cellular response, it is informative to have accurate assays that reveal not only average telomere lengths, but also the distribution of the longest and shortest telomeres in a given sample. Herein we report for the first time, the development of single telomere length analysis (STELA)—a PCR-based assay that amplifies multiple, individual telomeres— for Ustilago maydis, a basidiomycete fungus. Compared to the standard telomere Southern technique, STELA revealed a broader distribution of telomere size as well as the existence of relatively short telomeres in wild type cells. When applied to blm∆, a mutant thought to be defective in telomere replication, STELA revealed preferential loss of long telomeres, whose maintenance may thus be especially dependent upon efficient replication. In comparison to blm∆, the trt1∆ (telomerase null) mutant exhibited greater erosion of short telomeres, consistent with a special role for telomerase in re-lengthening extra-short telomeres. We also used STELA to characterize the 5’ ends of telomere C-strand, and found that in U. maydis, they terminate preferentially at selected nucleotide positions within the telomere repeat. Deleting trt1 altered the 5’-end distributions, suggesting that telomerase may directly or indirectly modulate C-strand 5’ end formation. These findings illustrate the utility of STELA as well as the strengths of U. maydis as a model system for telomere research.

The leishmaniases are worldwide neglected tropical diseases caused by parasitic protozoa of the Leishmania genus. Different stimuli induce Leishmania cell death, but the proteins involved remain poorly understood. Furthermore, confusion often appears between cell death and the cell survival process autophagy, whose phenotype is not clearly defined. In this article, we present a comprehensive and temporal analysis of the cellular events occurring during miltefosine-induced cell death and autophagy in L. major. We also provide a list of features in order to clearly identify apoptotic cells, autophagic cells and to distinguish both processes. Furthermore, we demonstrate that autophagy is followed by apoptosis in the absence of nutrients. Finally, we show that cells treated with the generic kinase inhibitor staurosporine express apoptotic as well as autophagic markers and therefore cannot be used as an apoptosis inducer in Leishmania. These descriptions lead to a better recognition and understanding of apoptosis and autophagy, enabling their targeting in the development of new anti-leishmanial drugs. These researches also make it possible to better understand these processes in general, through the study of an ancestral eukaryote.

Escherichia coli (E. coli) penetration of the blood–brain barrier (BBB) is the key step essential for the development of meningitis. In a recent paper (Nat Commun 9:2296), we identify Caspr1 as a host receptor for E. coli virulence factor IbeA to pave the way the penetration of bacteria through the BBB. Bacterial IbeA interacts with endothelial Caspr1 to trigger intracellular focal adhesion kinase activation, leading to E. coli internalization into the brain endothelial cells. Importantly, endothelial knockout of Caspr1 in mice significantly reduced E. coli crossing through the BBB. Based on the results that extracellular aa 203-355 of Caspr1 bind with IbeA, we tested the blocking effect of recombinant Caspr1(203-355) peptides in neonatal rat model of meningitis. The results showed that Caspr1(203-355) peptides effectively attenuated E. coli penetration into the brain during meningitis, indicating that Caspr1(203-355) peptides could be used to neutralize the virulent IbeA to prevent meningitis. We further found that E. coli can directly invade into hippocampal neurons causing apoptosis which required the interaction between bacterial IbeA and neuronal Caspr1. These findings demonstrate that E. coli hijack Caspr1 as a host receptor for penetration of BBB and invasion of hippocampal neurons, resulting in progression of meningitis.

Clostridium difficile, also known as Clostriodioides difficile, is a Gram positive, spore-forming bacterium and a leading cause of antibiotic-associated diarrhea in nosocomial environments. The key virulence factors of this pathogen are two toxins, toxin A and toxin B, released from the cells to the gut and causing colonic injury and inflammation. Although their mechanism of action is well known, the toxins A and B have no peptide signals and their secretion mechanisms involving the holin-like protein TcdE and autolysis are still under active investigation. Autolysis is primarily mediated by peptidoglycan hydrolases, an important group of enzymes that cleave covalent bonds in the cell wall peptidoglycan. Peptidoglycan hydrolases are essential for peptidoglycan remodeling but most of them also have the potential to lyse the cells under various conditions. In a recent report by Wydau-Dematteis et al. (MBio 9(3): e00648-18), we characterized a novel peptidoglycan hydrolase Cwp19 in C. difficile. Importantly, Cwp19 mediates toxins secretion in a glucose-dependent fashion suggesting a potential role in C. difficile pathogenesis. Peptidoglycan hydrolases are not very well characterized in C. difficile despite the important role of these enzymes in cell division and sporulation as shown in model organisms like Bacillus subtilis. In addition, these enzymes can be implicated in pathogenicity as exemplified by the release of pneumococcal virulence factors.