Domain shuffling of a highly mutable ligand-binding fold drives adhesin generation across the bacterial kingdom.

Bacterial fibrillar adhesins are specialized extracellular polypeptides that promote the attachment of bacteria to the surfaces of other cells or materials. Adhesin-mediated interactions are critical for the establishment and persistence of stable bacterial populations within diverse environmental niches and are important determinants of virulence. The fibronectin (Fn)-binding fibrillar adhesin CshA, and its paralogue CshB, play important roles in host colonization by the oral commensal and opportunistic pathogen Streptococcus gordonii. As paralogues are often catalysts for functional diversification, we have probed the early stages of structural and functional divergence in Csh proteins by determining the X-ray crystal structure of the CshB adhesive domain NR2 and characterizing its Fn-binding properties in vitro. Despite sharing a common fold, CshB_NR2 displays an ~1.7-fold reduction in Fn-binding affinity relative to CshA_NR2. This correlates with reduced electrostatic charge in the Fn-binding cleft. Complementary bioinformatic studies reveal that homologues of CshA/B_NR2 domains are widely distributed in both Gram-positive and Gram-negative bacteria, where they are found housed within functionally cryptic multi-domain polypeptides. Our findings are consistent with the classification of Csh adhesins and their relatives as members of the recently defined polymer adhesin domain (PAD) family of bacterial proteins.

Reciprocal best structure hits: using AlphaFold models to discover distant homologues.

Motivation: The conventional methods to detect homologous protein pairs use the comparison of protein sequences. But the sequences of two homologous proteins may diverge significantly and consequently may be undetectable by standard approaches. The release of the AlphaFold 2.0 software enables the prediction of highly accurate protein structures and opens many opportunities to advance our understanding of protein functions, including the detection of homologous protein structure pairs. Results: In this proof-of-concept work, we search for the closest homologous protein pairs using the structure models of five model organisms from the AlphaFold database. We compare the results with homologous protein pairs detected by their sequence similarity and show that the structural matching approach finds a similar set of results. In addition, we detect potential novel homologs solely with the structural matching approach, which can help to understand the function of uncharacterized proteins and make previously overlooked connections between well-characterized proteins. We also observe limitations of our implementation of the structure-based approach, particularly when handling highly disordered proteins or short protein structures. Our work shows that high accuracy protein structure models can be used to discover homologous protein pairs, and we expose areas for improvement of this structural matching approach. Availability and Implementation: Information to the discovered homologous protein pairs can be found at the following URL: https://doi.org/10.17863/CAM.87873. The code can be accessed here: https://github.com/VivianMonzon/Reciprocal_Best_Structure_Hits. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Bacterial retrons encode phage-defending tripartite toxin-antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA1 (msDNA). Despite decades of research on the biosynthesis of msDNA2, the function and physiological roles of retrons have remained unknown. Here we show that Retron-Sen2 of Salmonella enterica serovar Typhimurium encodes an accessory toxin protein, STM14_4640, which we renamed as RcaT. RcaT is neutralized by the reverse transcriptase-msDNA antitoxin complex, and becomes active upon perturbation of msDNA biosynthesis. The reverse transcriptase is required for binding to RcaT, and the msDNA is required for the antitoxin activity. The highly prevalent RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin-antitoxin system. To understand the physiological roles of such toxin-antitoxin systems, we developed toxin activation-inhibition conjugation (TAC-TIC), a high-throughput reverse genetics approach that identifies the molecular triggers and blockers of toxin-antitoxin systems. By applying TAC-TIC to Retron-Sen2, we identified multiple trigger and blocker proteins of phage origin. We demonstrate that phage-related triggers directly modify the msDNA, thereby activating RcaT and inhibiting bacterial growth. By contrast, prophage proteins circumvent retrons by directly blocking RcaT. Consistently, retron toxin-antitoxin systems act as abortive infection anti-phage defence systems, in line with recent reports3,4. Thus, RcaT retrons are tripartite DNA-regulated toxin-antitoxin systems, which use the reverse transcriptase-msDNA complex both as an antitoxin and as a sensor of phage protein activities.

Large-Scale Discovery of Microbial Fibrillar Adhesins and Identification of Novel Members of Adhesive Domain Families.

Fibrillar adhesins are bacterial cell surface proteins that mediate interactions with the environment, including host cells during colonization or other bacteria during biofilm formation. These proteins are characterized by a stalk that projects the adhesive domain closer to the binding target. Fibrillar adhesins evolve quickly and thus can be difficult to computationally identify, yet they represent an important component for understanding bacterium-host interactions. To detect novel fibrillar adhesins, we developed a random forest prediction approach based on common characteristics we identified for this protein class. We applied this approach to Firmicutes and Actinobacteria proteomes, yielding over 6,500 confidently predicted fibrillar adhesins. To verify the approach, we investigated predicted fibrillar adhesins that lacked a known adhesive domain. Based on these proteins, we identified 24 sequence clusters representing potential novel members of adhesive domain families. We used AlphaFold to verify that 15 clusters showed structural similarity to known adhesive domains, such as the TED domain. Overall, our study has made a significant contribution to the number of known fibrillar adhesins and has enabled us to identify novel members of adhesive domain families involved in bacterial pathogenesis. IMPORTANCE Fibrillar adhesins are a class of bacterial cell surface proteins that enable bacteria to interact with their environment. We developed a machine learning approach to identify fibrillar adhesins and applied this classification approach to the Firmicutes and Actinobacteria Reference Proteomes database. This method allowed us to detect a high number of novel fibrillar adhesins and also novel members of adhesive domain families. To confirm our predictions of these potential adhesin protein domains, we predicted their structure using the AlphaFold tool.

The effect of landscape and human settlement on the genetic differentiation and presence of Paragonimus species in Mesoamerica.

Foodborne diseases are a neglected research area, and despite the existence of many tools for diagnosis and genetic studies, very little is known about the effect of the landscape on the genetic diversity and presence of parasites. One of these foodborne disease is paragonimiasis, caused by trematodes of the genus Paragonimus, which is responsible for a high number of infections in humans and wild animals. The main Paragonimus sp reported in Mesoamerica is Paragonimus mexicanus, yet there are doubts about its correct identification as a unique species throughout the region. This, together with a lack of detailed knowledge about their ecology, evolution and differentiation, may complicate the implementation of control strategies across the Mesoamerican region. We had the goal of delimiting the species of P. mexicanus found throughout Mesoamerica and determining the effect of landscape and geology on the diversity and presence of the parasite. We found support for the delimitation of five genetic groups. The genetic differentiation among these groups was positively affected by elevation and the isolation of river basins, while the parasite's presence was affected negatively only by the presence of human settlements. These results suggest that areas with lower elevation, connected rivers basins, and an absence of human settlements have low genetic differentiation and high P. mexicanus presence, which may increase the risk of Paragonimus infection. These demonstrate the importance of accurate species delimitation and consideration of the effect of landscape on Paragonimus in the proposal of adequate control strategies. However, other landscape variables cannot be discarded, including temperature, rainfall regime, and spatial scale (local, landscape and regional). These additional variables were not explored here, and should be considered in future studies.

Folding the unfoldable: using AlphaFold to explore spurious proteins.

Motivation: The release of AlphaFold 2.0 has revolutionized our ability to determine protein structures from sequences. This tool also inadvertently opens up many unanticipated opportunities. In this article, we investigate the AntiFam resource, which contains 250 protein sequence families that we believe to be spurious protein translations. We would not expect proteins belonging to these families to fold into well-ordered globular structures. To test this hypothesis, we have attempted to computationally determine the structure of a representative sequence from all AntiFam 6.0 families. Results: Although the large majority of families showed no evidence of globular structure, we have identified one example for which a globular structure is predicted. Proteins in this AntiFam entry indeed seem likely to be bona fide proteins, based on additional considerations, and thus AlphaFold provides a useful quality control for the AntiFam database. Conversely, known spurious proteins offer useful set of quality controls for AlphaFold. We have identified a trend that the mean structure prediction confidence score pLDDT is higher for shorter sequences. Of the 131 AntiFam representative sequences <100 amino acids in length, AlphaFold predicts a mean pLDDT of 80 or greater for six of them. Thus, particular care should be taken when applying AlphaFold to short protein sequences. Availability and implementation: The AlphaFold predictions for representative sequences can be found at the following URL: https://drive.google.com/drive/folders/1u9OocRIAabGQn56GljoG1JTDAxjkY1ro. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Caracterización de pacientes en aislamiento por COVID-19 en la Universidad de Ciencias Médicas. Cienfuegos, 2021 / Characterization of patients in isolation due to COVID-19 at the University of Medical Sciences. Cienfuegos, 2021

RESUMEN Fundamento: la pandemia de COVID-19 es la crisis sanitaria que define nuestro tiempo y que ha puesto a prueba los sistemas de salud en todo el mundo. Objetivo describir las características clínico epidemiológicas de pacientes sospechosos de COVID-19, en aislamiento durante el primer cuatrimestre del 2021. Métodos estudio observacional, descriptivo, transversal. El universo de estudio fue de 2 631 pacientes contactos y sospechosos de la enfermedad ingresados en dos centros de aislamiento en la Universidad de Ciencias Médicas de Cienfuegos, desde enero hasta abril 2021. Se analizaron las variables: edad, sexo, ocupación, municipio de residencia, antecedenetes epidemiológicos, comorbilidades y confirmación por la prueba de cadena de polimerasa en tiempo real. Se confeccionó una encuesta epidemiológica e historia clínica individual durante el aislamiento. Resultados: el 78,3 % de los pacientes en aislamiento fueron contacto de casos confirmados, los pacientes en edad laboral representaron el mayor porciento, con predominio del sexo femenino. El municipio de Cienfuegos aportó el mayor número de pacientes. El 59,2 % de estos tenía al menos una comorbilidad, más del 50 % eran hipertensos. El 8 % de los sospechosos fueron positivos a la COVID-19. Conclusiones el aislamiento de contacto y sospechosos como medida de contención de la pandemia representa una estrategia de impacto. Conocer las características de los pacientes aislados sirve como línea de base para el desarrollo de nuevas medidas sanitarias.

ABSTRACT Background: The COVID-19 pandemic is the defining health crisis of our time that has put health systems to the test around the world. Objective: to describe the clinical epidemiological characteristics of patients in isolation, during the first four months of 2021. Methods: observational, descriptive, cross-sectional study. The study universe consisted of 2 631 patients who were contacts and suspected of the disease admitted to two isolation centers at the University of Medical Sciences of Cienfuegos, from January to April 2021. The variables were analyzed: age, sex, occupation, municipality of residence, epidemiological antecedents, comorbidities and confirmation by the real-time polymerase chain test. An epidemiological survey and individual medical history were prepared during isolation. Results: 78.3% of the patients in isolation were contacts of confirmed cases, the patients of working age represented the highest percentage, with a predominance of the female sex. The municipality of Cienfuegos contributed the largest number of patients. 59.2% of these had at least one comorbidity, more than 50% were hypertensive. 8% of the suspects were positive for COVID-19. Conclusions: the isolation of contact and suspects as a measure to contain the pandemic represents an impact strategy. Knowing the characteristics of isolated patients serves as a baseline for the development of new health measures.

Discovery of fibrillar adhesins across bacterial species.

BACKGROUND: Fibrillar adhesins are long multidomain proteins that form filamentous structures at the cell surface of bacteria. They are an important yet understudied class of proteins composed of adhesive and stalk domains that mediate interactions of bacteria with their environment. This study aims to characterize fibrillar adhesins in a wide range of bacterial phyla and to identify new fibrillar adhesin-like proteins to improve our understanding of host-bacteria interactions. RESULTS: Through careful literature and computational searches, we identified 82 stalk and 27 adhesive domain families in fibrillar adhesins. Based on the presence of these domains in the UniProt Reference Proteomes database, we identified and analysed 3,542 fibrillar adhesin-like proteins across species of the most common bacterial phyla. We further enumerate the adhesive and stalk domain combinations found in nature and demonstrate that fibrillar adhesins have complex and variable domain architectures, which differ across species. By analysing the domain architecture of fibrillar adhesins, we show that in Gram positive bacteria, adhesive domains are mostly positioned at the N-terminus and cell surface anchors at the C-terminus of the protein, while their positions are more variable in Gram negative bacteria. We provide an open repository of fibrillar adhesin-like proteins and domains to enable further studies of this class of bacterial surface proteins. CONCLUSION: This study provides a domain-based characterization of fibrillar adhesins and demonstrates that they are widely found in species across the main bacterial phyla. We have discovered numerous novel fibrillar adhesins and improved our understanding of pathogenic adhesion and invasion mechanisms.

The small DUF1127 protein CcaF1 from Rhodobacter sphaeroides is an RNA-binding protein involved in sRNA maturation and RNA turnover.

Many different protein domains are conserved among numerous species, but their function remains obscure. Proteins with DUF1127 domains number >17 000 in current databases, but a biological function has not yet been assigned to any of them. They are mostly found in alpha- and gammaproteobacteria, some of them plant and animal pathogens, symbionts or species used in industrial applications. Bioinformatic analyses revealed similarity of the DUF1127 domain of bacterial proteins to the RNA binding domain of eukaryotic Smaug proteins that are involved in RNA turnover and have a role in development from Drosophila to mammals. This study demonstrates that the 71 amino acid DUF1127 protein CcaF1 from the alphaproteobacterium Rhodobacter sphaeroides participates in maturation of the CcsR sRNAs that are processed from the 3' UTR of the ccaF mRNA and have a role in the oxidative stress defense. CcaF1 binds to many cellular RNAs of different type, several mRNAs with a function in cysteine / methionine / sulfur metabolism. It affects the stability of the CcsR RNAs and other non-coding RNAs and mRNAs. Thus, the widely distributed DUF1127 domain can mediate RNA-binding, affect stability of its binding partners and consequently modulate the bacterial transcriptome, thereby influencing different physiological processes.

Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing.

Bacterial small noncoding RNAs (sRNAs) play posttranscriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV cross-linking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the Pseudomonas aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5' untranslated regions (UTRs) and sRNAs and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that the association of sRNAs with Hfq is primarily a function of their expression levels, strongly supporting the notion that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of posttranscriptional regulation in P. aeruginosa IMPORTANCE The Gram-negative bacterium P. aeruginosa is ubiquitously distributed in diverse environments and can cause severe biofilm-related infections in at-risk individuals. Although the presence of a large number of putative sRNAs and widely conserved RNA chaperones in this bacterium implies the importance of posttranscriptional regulatory networks for environmental fluctuations, limited information is available regarding the global role of RNA chaperones such as Hfq in the P. aeruginosa transcriptome, especially under different environmental conditions. Here, we characterize Hfq-dependent differences in gene expression and biological processes in two physiological states: the planktonic and biofilm forms. A combinatorial comparative CLIP-seq and total RNA-seq approach uncovered condition-dependent association of RNAs with Hfq in vivo and expands the potential direct regulatory targets of Hfq in the P. aeruginosa transcriptome.

Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq.

Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5'-UTRs and with very long 3'-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3'-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.

The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978.

We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3' end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of -10 and -35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as 'AcinetoCom' (http://bioinf.gen.tcd.ie/acinetocom/).

Coinfección VIH-hepatitis B y C en la provincia de Cienfuegos / HIV/hepatitis B and C co-infection in Cienfuegos province

Objetivo: identificar la prevalencia de la infección por los virus de la hepatitis B (VHB) y C (VHC) en individuos infectados por VIH en la provincia de Cienfuegos. Métodos: se realizó un estudio de corte transversal hasta el mes del abril de 2005 y se analizaron variables clínicas y el recuento de linfocitos TCD4+ (LTCD4+). Resultados: la prevalencia de la infección por el VHB resultó 14,4 por ciento y por el VHC 20 por ciento. La frecuencia del SIDA fue superior en los pacientes con hepatitis B (77,8 por ciento) respecto a los infectados por el VHC (28,6 por ciento). El porcentaje de casos con menos de 500 LTCD4+ resultó superior en los coinfectados por el VHC (57,4 por ciento); 4 enfermos (14,8 por ciento) tenían realizado estudio histológico hepático. Conclusión: la coinfección de los VHB/VHC y el VIH es un problema frecuente en la provincia, sin embargo, se desconocen aspectos clínicos y epidemiológicos que ameritan nuevos estudios.

Objective: To identify the prevalence of hepatitis B and C virus infections (HVB) and (HVC) in individuals infected by HIV in Cienfuegos province. Methods: A cross-sectional study was performed till April 2005 in which clinical variables and TCD4+ lymphocyte count were analyzed. Results: the prevalence of HVB infection was 14,4 percent and of HVC was 20 percent. AIDS frequency was higher in patients suffering from hepatitis B (77,8 percent) than in those with hepatitis C (28,6 percent). Percentage of cases under 500 TCD4+ count was higher in patients coinfested with HVC (57,4 percent); also 4 patients (14,8 percent) had been performed a hepatic histological study. Conclusion: HVB/HVC co-infection and HIV is a common problem in the province; however, clinical and epidemiological aspects that are yet unknown call for new studies.

Coinfección VIH-hepatitis B y C en la provincia de Cienfuegos / HIV/hepatitis B and C co-infection in Cienfuegos province

Objetivo: identificar la prevalencia de la infección por los virus de la hepatitis B (VHB) y C (VHC) en individuos infectados por VIH en la provincia de Cienfuegos. Métodos: se realizó un estudio de corte transversal hasta el mes del abril de 2005 y se analizaron variables clínicas y el recuento de linfocitos TCD4+ (LTCD4+). Resultados: la prevalencia de la infección por el VHB resultó 14,4 por ciento y por el VHC 20 por ciento. La frecuencia del SIDA fue superior en los pacientes con hepatitis B (77,8 por ciento) respecto a los infectados por el VHC (28,6 por ciento). El porcentaje de casos con menos de 500 LTCD4+ resultó superior en los coinfectados por el VHC (57,4 por ciento); 4 enfermos (14,8 por ciento) tenían realizado estudio histológico hepático. Conclusión: la coinfección de los VHB/VHC y el VIH es un problema frecuente en la provincia, sin embargo, se desconocen aspectos clínicos y epidemiológicos que ameritan nuevos estudios(AU)

Objective: To identify the prevalence of hepatitis B and C virus infections (HVB) and (HVC) in individuals infected by HIV in Cienfuegos province. Methods: A cross-sectional study was performed till April 2005 in which clinical variables and TCD4+ lymphocyte count were analyzed. Results: the prevalence of HVB infection was 14,4 percent and of HVC was 20 percent. AIDS frequency was higher in patients suffering from hepatitis B (77,8 percent) than in those with hepatitis C (28,6 percent). Percentage of cases under 500 TCD4+ count was higher in patients coinfested with HVC (57,4 percent); also 4 patients (14,8 percent) had been performed a hepatic histological study. Conclusion: HVB/HVC co-infection and HIV is a common problem in the province; however, clinical and epidemiological aspects that are yet unknown call for new studies(AU)