Sensing of intracellular Hcp levels controls T6SS expression in Vibrio cholerae.

The type 6 secretion system (T6SS) is a bacterial weapon broadly distributed in gram-negative bacteria and used to kill competitors and predators. Featuring a long and double-tubular structure, this molecular machine is energetically costly to produce and thus is likely subject to diverse regulation strategies that are largely ill defined. In this study, we report a quantity-sensing control of the T6SS that down-regulates the expression of secreted components when they accumulate in the cytosol due to T6SS inactivation. Using Vibrio cholerae strains that constitutively express an active T6SS, we demonstrate that mRNA levels of secreted components, including the inner-tube protein component Hcp, were down-regulated in T6SS structural gene mutants while expression of the main structural genes remained unchanged. Deletion of both hcp gene copies restored expression from their promoters, while Hcp overexpression negatively impacted expression. We show that Hcp directly interacts with the RpoN-dependent T6SS regulator VasH, and deleting the N-terminal regulator domain of VasH abolishes this interaction as well as the expression difference of hcp operons between T6SS-active and inactive strains. We find that negative regulation of hcp also occurs in other V. cholerae strains and the pathogens Aeromonas dhakensis and Pseudomonas aeruginosa This Hcp-dependent sensing control is likely an important energy-conserving mechanism that enables T6SS-encoding organisms to quickly adjust T6SS expression and prevent wasteful build-up of its major secreted components in the absence of their efficient export out of the bacterial cell.

Genome-Wide Ribosome Profiling of the Plasmodium falciparum Intraerythrocytic Developmental Cycle.

Monitoring whole-genome translation and mRNA ribosome occupancy in vivo using ribosome profiling has proven to be a powerful tool for discovery of gene expression regulation, mechanisms of translation, and new open reading frames, in a wide range of different cell types in different organisms. Here we describe its application to the malaria parasite, Plasmodium falciparum. We present methods for intact polysome purification from parasite cultures, polysome digestion, monosome purification, ribosome footprint nucleic acid extraction, and Illumina library preparation.

Transcriptional Silencing by TsrA in the Evolution of Pathogenic Vibrio cholerae Biotypes.

Vibrio cholerae is a globally important pathogen responsible for the severe epidemic diarrheal disease called cholera. The current and ongoing seventh pandemic of cholera is caused by El Tor strains, which have completely replaced the sixth-pandemic classical strains of V. cholerae To successfully establish infection and disseminate to new victims, V. cholerae relies on key virulence factors encoded on horizontally acquired genetic elements. The expression of these factors relies on the regulatory architecture that coordinates the timely expression of virulence determinants during host infection. Here, we apply transcriptomics and structural modeling to understand how type VI secretion system regulator A (TsrA) affects gene expression in both the classical and El Tor biotypes of V. cholerae We find that TsrA acts as a negative regulator of V. cholerae virulence genes encoded on horizontally acquired genetic elements. The TsrA regulon comprises genes encoding cholera toxin (CT), the toxin-coregulated pilus (TCP), and the type VI secretion system (T6SS), as well as genes involved in biofilm formation. The majority of the TsrA regulon is carried on horizontally acquired AT-rich genetic islands whose loss or acquisition could be directly ascribed to the differences between the classical and El Tor strains studied. Our modeling predicts that the TsrA protein is a structural homolog of the histone-like nucleoid structuring protein (H-NS) oligomerization domain and is likely capable of forming higher-order superhelical structures, potentially with DNA. These findings describe how TsrA can integrate into the intricate V. cholerae virulence gene expression program, controlling gene expression through transcriptional silencing.IMPORTANCE Pathogenic Vibrio cholerae strains express multiple virulence factors that are encoded by bacteriophage and chromosomal islands. These include cholera toxin and the intestinal colonization pilus called the toxin-coregulated pilus, which are essential for causing severe disease in humans. However, it is presently unclear how the expression of these horizontally acquired accessory virulence genes can be efficiently integrated with preexisting transcriptional programs that are presumably fine-tuned for optimal expression in V. cholerae before its conversion to a human pathogen. Here, we report the role of a transcriptional regulator (TsrA) in silencing horizontally acquired genes encoding important virulence factors. We propose that this factor could be critical to the efficient acquisition of accessory virulence genes by silencing their expression until other signals trigger their transcriptional activation within the host.

Microbiota-targeted maternal antibodies protect neonates from enteric infection.

Although maternal antibodies protect newborn babies from infection1,2, little is known about how protective antibodies are induced without prior pathogen exposure. Here we show that neonatal mice that lack the capacity to produce IgG are protected from infection with the enteric pathogen enterotoxigenic Escherichia coli by maternal natural IgG antibodies against the maternal microbiota when antibodies are delivered either across the placenta or through breast milk. By challenging pups that were fostered by either maternal antibody-sufficient or antibody-deficient dams, we found that IgG derived from breast milk was crucial for protection against mucosal disease induced by enterotoxigenic E. coli. IgG also provides protection against systemic infection by E. coli. Pups used the neonatal Fc receptor to transfer IgG from milk into serum. The maternal commensal microbiota can induce antibodies that recognize antigens expressed by enterotoxigenic E. coli and other Enterobacteriaceae species. Induction of maternal antibodies against a commensal Pantoea species confers protection against enterotoxigenic E. coli in pups. This role of the microbiota in eliciting protective antibodies to a specific neonatal pathogen represents an important host defence mechanism against infection in neonates.

Analysis of lipoprotein transport depletion in Vibrio cholerae using CRISPRi.

Genes necessary for the survival or reproduction of a cell are an attractive class of antibiotic targets. Studying essential genes by classical genetics, however, is inherently problematic because it is impossible to knock them out. Here, we screened a set of predicted essential genes for growth inhibition using CRISPR-interference (CRISPRi) knockdown in the human pathogen Vibrio cholerae We demonstrate that CRISPRi knockdown of 37 predicted essential genes inhibits V. cholerae viability, thus validating the products of these genes as potential drug target candidates. V. cholerae was particularly vulnerable to lethal inhibition of the system for lipoprotein transport (Lol), a central hub for directing lipoproteins from the inner to the outer membrane (OM), with many of these lipoproteins coordinating their own essential processes. Lol depletion makes cells prone to plasmolysis and elaborate membrane reorganization, during which the periplasm extrudes into a mega outer membrane vesicle or "MOMV" encased by OM which dynamically emerges specifically at plasmolysis sites. Our work identifies the Lol system as an ideal drug target, whose inhibition could deplete gram-negative bacteria of numerous proteins that reside in the periplasm.

Antagonism toward the intestinal microbiota and its effect on Vibrio cholerae virulence.

The bacterial type VI secretion system (T6SS) is a nanomachine that delivers toxic effector proteins into target cells, killing them. In mice, we found that the Vibrio cholerae T6SS attacks members of the host commensal microbiota in vivo, facilitating the pathogen's colonization of the gut. This microbial antagonistic interaction drives measurable changes in the pathogenicity of V. cholerae through enhanced intestinal colonization, expression of bacterial virulence genes, and activation of host innate immune genes. Because ablation of mouse commensals by this enteric pathogen correlated with more severe diarrheal symptoms, we conclude that antagonism toward the gut microbiota could improve the fitness of V. cholerae as a pathogen by elevating its transmission to new susceptible hosts.

Genome-wide regulatory dynamics of translation in the Plasmodium falciparum asexual blood stages.

The characterization of the transcriptome and proteome of Plasmodium falciparum has been a tremendous resource for the understanding of the molecular physiology of this parasite. However, the translational dynamics that link steady-state mRNA with protein levels are not well understood. In this study, we bridge this disconnect by measuring genome-wide translation using ribosome profiling, through five stages of the P. falciparum blood phase developmental cycle. Our findings show that transcription and translation are tightly coupled, with overt translational control occurring for less than 10% of the transcriptome. Translationally regulated genes are predominantly associated with merozoite egress functions. We systematically define mRNA 5' leader sequences, and 3' UTRs, as well as antisense transcripts, along with ribosome occupancy for each, and establish that accumulation of ribosomes on 5' leaders is a common transcript feature. This work represents the highest resolution and broadest portrait of gene expression and translation to date for this medically important parasite.

Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum.

Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum.

Trypanosoma cruzi: analysis of the complete PUF RNA-binding protein family.

The members of the PUF family of RNA-binding proteins regulate the fate of mRNAs by binding to their 3'UTR sequence elements in eukaryotes. In trypanosomes, for which gene expression is polycistronic and controlled almost exclusively by post-transcriptional processes, PUF proteins could play a crucial role. We report here the complete analysis of the PUF protein family of Trypanosoma cruzi composed of 10 members. In silico analysis predicts the existence of at least three major groups within the T. cruzi family, based on their putative binding specificity. Using yeast three hybrid assays, we tested some of these predictions for TcPUF1, TcPUF3, TcPUF5, and TcPUF8 as representatives of these groups. Data mining of the T. cruzi genome led us to describe putative binding targets for the TcPUFs of the most conserved group, TcPUF1 and TcPUF2. The targets include genes for mitochondrial proteins and protein kinases. Finally, immunolocalization experiments showed that TcPUF1 is localized in multiple discrete foci in the cytoplasm supporting its proposed function.

Protein interactions within the TcZFP zinc finger family members of Trypanosoma cruzi: implications for their functions.

The small zinc finger proteins tbZFP1 and tbZFP2 have been implicated in the control of Trypanosoma brucei differentiation to the procyclic form. Here, we report that the complete ZFP family in Trypanosoma cruzi is composed by four members, ZFP1A and B, and ZFP2A and B. ZFP1B is a paralog specific gene restricted to T. cruzi, while the ZFP2A and B paralogs diverged prior to the trypanosomatid lineage separation. Moreover, we demonstrate that TcZFP1 and TcZFP2 members interact with each other and that this interaction is mediated by a WW domain in TcZFP2. Also, TcZFP2B strongly homodimerizes by a glycine rich region absent in TcZFP2A. We propose a model to discuss the relevance of these protein-protein interactions in terms of the functions of these proteins.