Biology subjects

Gonzalez, D.

Publications and source records attributed to Gonzalez, D..

42 records · Page 3Linked to original sources

bioRxiv2021

The DEAD-box RNA helicases RhlE2 is a global regulator of Pseudomonas aeruginosa lifestyle and pathogenesis

The RhlE DEAD-box RNA helicase protein family is widespread among Proteobacteria, but it is the least understood due to the lack of a clear biological function. Here, we study the two RhlE homologs present in the opportunistic pathogen Pseudomonas aeruginosa. RhlE1 and RhlE2 diverged during P. aeruginosa evolution; our data indicate that this resulted in a non-redundant biological role, a distinct molecular action and an enzymatic activity differentially stimulated by RNA. Whereas RhlE1 is specifically necessary for bacteria growth in cold, we show that RhlE2 acts as global post-transcriptional regulator, affecting the level of hundreds of cellular transcripts and multiple functionalities indispensable not only for P. aeruginosa environmental adaptation, but also for its virulence. The global action of RhlE2 relies on a unique C-terminal extension, which establishes an RNA-dependent interaction with the RNase E endonuclease and the cellular RNA degradation machinery.

microbiology↗

bioRxiv2020

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community1,2. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs3-8. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins9,10. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in their ecology.

microbiology↗

bioRxiv2019

Targeted Sequencing Workflows for Comprehensive Drug Resistance Profiling of Mycobacterium tuberculosis cultures using Illumina MiSeq and Nanopore MinION: Comparison of analytical and diagnostic performance, turnaround time and cost

The emergence of Mycobacterium tuberculosis strains with complex drug resistance profiles necessitates a rapid and extensive drug susceptibility test for comprehensive guidance of patient treatment. Here, we developed two targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the prediction of drug resistance in M. tuberculosis towards 12 anti-tuberculous agents.\n\nA total of 163 M. tuberculosis cultured isolates collected from Hong Kong and Ethiopia were subjected to a multiplex PCR for simultaneous amplification of 19 drug-resistance associated genetic regions. The amplicons were then barcoded and sequenced in parallel on MiSeq and MinION in respective batch sizes of 24 and 12 samples. Both platforms successfully sequenced all samples with average depths of coverage of 1,127x and 1,649x respectively. Utilizing a self-developed Web-based bioinformatics pipeline, Bacteriochek-TB, for variant analysis, we found that the MiSeq and MinION result could achieve 100% agreement if variants with an allele frequency of <40% reported by MinION were excluded. For drug resistance prediction, both workflows achieved an average sensitivity of 94.8% and specificity of 98.0% when compared with phenotypic drug susceptibility test. The turnaround times for the MiSeq and MinION workflows were 38 and 15 hours, facilitating the delivery of treatment guidance at least 17-18 days earlier than pDST respectively. The higher cost per sample on the MinION platform (US$71.56) versus the MiSeq platform (US$67.83) was attributed to differences in batching capabilities.\n\nOur study demonstrated the interchangeability of MiSeq and MinION sequencing workflows for generation of accurate and actionable results for the treatment of tuberculosis.\n\nImportanceTB therapy involving different combinations of antibiotics have been introduced to address the issue of drug resistance. However, this practice has led to increasing numbers of M. tuberculosis with complex drug resistance profiles. Molecular assays for rapid and comprehensive drug resistance profiling of M. tuberculosis are lacking.\n\nHere, we described targeted-sequencing workflows based on Illumina MiSeq and Nanopore MinION for the detection of drug resistance mutations scattered across 19 genetic regions in M. tuberculosis. A bioinformatics pipeline was also developed to translate raw datasets into clinician-friendly reports that provide comprehensive genetic information for the prediction of drug resistance towards 12 antibiotics.\n\nThis is the first study to evaluate and compare the uses of Illumina and Nanopore platforms for diagnosis of drug-resistant tuberculosis. Remarkably, our diagnostic strategy is compatible with different sequencing platforms that can be applied in diagnostic centres with different levels of throughput and financial support for TB diagnosis.

microbiology↗

bioRxiv2019

The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter

Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor {sigma}70. The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where {sigma}70 localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically,{Delta} cdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes. Notably, we find that{Delta} cdnL cells are severely limited in glutamate synthesis, rendering them auxotrophic for that amino acid. Moreover,{Delta} cdnL cells produce low amounts of the cell wall precursor lipid II, and{Delta} cdnL is synthetic lethal with other genetic perturbations that limit lipid II production.{Delta} cdnL cells also have aberrant localization of MreB and CtpS, cytoskeletal proteins required for maintaining proper cell width and curvature. Interestingly, the localization of CtpS is dependent on availability of CTP, which is predicted to be low in{Delta} cdnL cells. Our findings implicate CdnL as a global regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and through metabolite-mediated changes in localization of cytoskeletal regulators of cell shape.

microbiology↗

bioRxiv2018

EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

The mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired with in vitro cell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9a in vivo and in vitro to elicit {beta}-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCs in vitro.\n\nHighlightsO_LIAn in vitro signaling screen identifies Fzd9b as a Wnt9a-specific receptor.\nC_LIO_LIFzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.\nC_LIO_LIWNT9A and FZD9 are required for HSPC derivation from human pluripotent cells in vitro.\nC_LIO_LIEGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.\nC_LI

developmental biology↗

bioRxiv2018

The Relationship Between the Cervical Microbiome, HIV Status, and Pre-Cancerous Lesions

Nearly all cervical cancers are causally associated with Human Papillomavirus (HPV). The burden of HPV-associated dysplasias in Sub-Saharan Africa is influenced by HIV. To investigate the role of the bacterial microbiome in cervical dysplasia, cytobrush samples were collected directly from cervical lesions of 144 Tanzanian women. The V4 hypervariable region of the 16S rRNA gene was amplified and deep-sequenced. Alpha diversity metrics; Chao1, PD whole tree, and operational taxonomic Unit (OTU) estimates, displayed significantly higher bacterial richness in HIV positive patients (P = 0.01) than in HIV negative patients. Within HIV positive patients, there was higher bacterial richness in patients with high grade squamous intraepithelial lesions (HSIL; P = 0.13) than those without lesions. The most abundant OTUs associated with high-grade squamous intraepitheilal lesions (HSIL) were Mycoplasmatales, Pseudomonadales, and Staphylococcus. We suggest that a chronic mycoplasma infection of the cervix can contribute to HPV-dependent dysplasia by sustained inflammatory signals.

cancer biology↗