Streptococcus pneumoniae: a Plethora of Temperate Bacteriophages With a Role in Host Genome Rearrangement

Bacteriophages (phages) are viruses that infect bacteria. They are the most abundant biological entity on Earth (current estimates suggest there to be perhaps 1031 particles) and are found nearly everywhere. Temperate phages can integrate into the chromosome of their host, and prophages have been found in abundance in sequenced bacterial genomes. Prophages may modulate the virulence of their host in different ways, e.g., by the secretion of phage-encoded toxins or by mediating bacterial infectivity. Some 70% of Streptococcus pneumoniae (the pneumococcus)-a frequent cause of otitis media, pneumonia, bacteremia and meningitis-isolates harbor one or more prophages. In the present study, over 4000 S. pneumoniae genomes were examined for the presence of prophages, and nearly 90% were found to contain at least one prophage, either defective (47%) or present in full (43%). More than 7000 complete putative integrases, either of the tyrosine (6243) or serine (957) families, and 1210 full-sized endolysins (among them 1180 enzymes corresponding to 318 amino acid-long N-acetylmuramoyl-L-alanine amidases [LytAPPH]) were found. Based on their integration site, 26 different pneumococcal prophage groups were documented. Prophages coding for tRNAs, putative virulence factors and different methyltransferases were also detected. The members of one group of diverse prophages (PPH090) were found to integrate into the 3' end of the host lytASpn gene encoding the major S. pneumoniae autolysin without disrupting it. The great similarity of the lytASpn and lytA PPH genes (85-92% identity) allowed them to recombine, via an apparent integrase-independent mechanism, to produce different DNA rearrangements within the pneumococcal chromosome. This study provides a complete dataset that can be used to further analyze pneumococcal prophages, their evolutionary relationships, and their role in the pathogenesis of pneumococcal disease.This research was supported by grants MPY 509/19 from the Instituto de Salud Carlos III (ISCIII) and SAF2017-88664-R from the Spanish Ministerio de Economía, Industria y Competitividad (MEICOM). The Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) is an initiative of the Instituto de Salud Carlos III (ISCIII). AM-G is the recipient of a Miguel Servet contract by the ISCIII.S

Computational characterization of the peptidome in transporter associated with antigen processing (TAP)-deficient cells

The transporter associated with antigen processing (TAP) is a key element of the major histocompatibility complex (MHC) class I antigen processing and presentation pathway. Nonfunctional TAP complexes impair the translocation of cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen. This drastic reduction in the available peptide repertoire leads to a significant decrease in MHC class I cell surface expression. Using mass spectrometry, different studies have analyzed the cellular MHC class I ligandome from TAP-deficient cells, but the analysis of the parental proteins, the source of these ligands, still deserves an in-depth analysis. In the present report, several bioinformatics protocols were applied to investigate the nature of parental proteins for the previously identified TAP-independent MHC class I ligands. Antigen processing in TAP-deficient cells mainly focused on small, abundant or highly integral transmembrane proteins of the cellular proteome. This process involved abundant proteins of the central RNA metabolism. In addition, TAP-independent ligands were preferentially cleaved from the N- and C-terminal ends with respect to the central regions of the parental proteins. The abundance of glycine, proline and aromatic residues in the C-terminal sequences from TAP-independently processed proteins allows the accessibility and specificity required for the proteolytic activities that generates the TAP-independent ligandome. This limited proteolytic activity towards a set of preferred proteins in a TAP-negative environment would therefore suffice to promote the survival of TAP-deficient individuals.This work was supported by the Spanish Ministry of Economy (MINECO/FEDER) grant SAF2014-58052 and Acción Estratégica en Salud 2019 to DL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptS

The Versatility of Opportunistic Infections Caused by Gemella Isolates Is Supported by the Carriage of Virulence Factors From Multiple Origins

The molecular basis of the pathogenesis of the opportunistic invasive infections caused by isolates of the Gemella genus remains largely unknown. Moreover, inconsistencies in the current species assignation were detected after genome-level comparison of 16 public Gemella isolates. A literature search detected that, between the two most pathogenic species, Gemella morbillorum causes about twice the number of cases compared to Gemella haemolysans. These two species shared their mean diseases - sepsis and endocarditis - but differed in causing other syndromes. A number of well-known virulence factors were harbored by all species, such as a manganese transport/adhesin sharing 83% identity from oral endocarditis-causing streptococci. Likewise, all Gemellae carried the genes required for incorporating phosphorylcholine into their cell walls and encoded some choline-binding proteins. In contrast, other proteins were species-specific, which may justify the known epidemiological differences. G. haemolysans, but not G. morbillorum, harbor a gene cluster potentially encoding a polysaccharidic capsule. Species-specific surface determinants also included Rib and MucBP repeats, hemoglobin-binding NEAT domains, peptidases of C5a complement factor and domains that recognize extracellular matrix molecules exposed in damaged heart valves, such as collagen and fibronectin. Surface virulence determinants were associated with several taxonomically dispersed opportunistic genera of the oral microbiota, such as Granulicatella, Parvimonas, and Streptococcus, suggesting the existence of a horizontally transferrable gene reservoir in the oral environment, likely facilitated by close proximity in biofilms and ultimately linked to endocarditis. The identification of the Gemella virulence pool should be implemented in whole genome-based protocols to rationally predict the pathogenic potential in ongoing clinical infections caused by these poorly known bacterial pathogens.This research was supported by grants MPY 509/19 from the Instituto de Salud Carlos III (ISCIII) and SAF2017-88664-R from the Spanish Ministerio de Economía, Industria y Competitividad (MEICOM). The Centro de Investigación Biomédica en Red de Enfermedades Respiratorias (CIBERES) is an initiative of the ISCIII. AM-G is the recipient of a Miguel Servet contract by the ISCIII.S

The challenges of the genome-based identification of antifungal resistance in the clinical routine

The increasing number of chronic and life-threatening infections caused by antimicrobial resistant fungal isolates is of critical concern. Low DNA sequencing cost may facilitate the identification of the genomic profile leading to resistance, the resistome, to rationally optimize the design of antifungal therapies. However, compared to bacteria, initiatives for resistome detection in eukaryotic pathogens are underdeveloped. Firstly, reported mutations in antifungal targets leading to reduced susceptibility must be extensively collected from the literature to generate comprehensive databases. This information should be complemented with specific laboratory screenings to detect the highest number possible of relevant genetic changes in primary targets and associations between resistance and other genomic markers. Strikingly, some drug resistant strains experience high-level genetic changes such as ploidy variation as much as duplications and reorganizations of specific chromosomes. Such variations involve allelic dominance, gene dosage increments and target expression regime effects that should be explicitly parameterized in antifungal resistome prediction algorithms. Clinical data indicate that predictors need to consider the precise pathogen species and drug levels of detail, instead of just genus and drug class. The concomitant needs for mutation accuracy and assembly quality assurance suggest hybrid sequencing approaches involving third-generation methods will be utilized. Moreover, fatal fast infections, like fungemia and meningitis, will further require both sequencing and analysis facilities are available in-house. Altogether, the complex nature of antifungal resistance demands extensive sequencing, data acquisition and processing, bioinformatic analysis pipelines, and standard protocols to be accomplished prior to genome-based protocols are applied in the clinical setting.This study was supported by the Acción Estratégica en Salud from Fondo de Investigaciones Sanitarias, ISCIII, grants MPY 509/19 and PI20CIII/00043.S

Identification of Promoter Region Markers Associated With Altered Expression of Resistance-Nodulation-Division Antibiotic Efflux Pumps in Acinetobacter baumannii

Genetic alterations leading to the constitutive upregulation of specific efflux pumps contribute to antibacterial resistance in multidrug resistant bacteria. The identification of such resistance markers remains one of the most challenging tasks of genome-level resistance predictors. In this study, 487 non-redundant genetic events were identified in upstream zones of three operons coding for resistance-nodulation-division (RND) efflux pumps of 4,130 Acinetobacter baumannii isolates. These events included insertion sequences, small indels, and single nucleotide polymorphisms. In some cases, alterations explicitly modified the expression motifs described for these operons, such as the promoter boxes, operators, and Shine-Dalgarno sequences. In addition, changes in DNA curvature and mRNA secondary structures, which are structural elements that regulate expression, were also calculated. According to their influence on RND upregulation, the catalog of upstream modifications were associated with "experimentally verified," "presumed," and "probably irrelevant" degrees of certainty. For experimental verification, DNA of upstream sequences independently carrying selected markers, three for each RND operon, were fused to a luciferase reporter plasmid system. Five out of the nine selected markers tested showed significant increases in expression with respect to the wild-type sequence control. In particular, a 25-fold expression increase was observed with the ISAba1 insertion sequence upstream the adeABC pump. Next, overexpression of each of the three multi-specific RND pumps was linked to their respective antibacterial substrates by a deep A. baumannii literature screen. Consequently, a data flow framework was then developed to link genomic upregulatory RND determinants to potential antibiotic resistance. Assignment of potential increases in minimal inhibitory concentrations at the "experimentally verified" level was permitted for 42 isolates to 7-8 unrelated antibacterial agents including tigecycline, which is overlooked by conventional resistome predictors. Thus, our protocol may represent a time-saving filter step prior to laborious confirmation experiments for efflux-driven resistance. Altogether, a computational-experimental pipeline containing all components required for identifying the upstream regulatory resistome is proposed. This schema may provide the foundational stone for the elaboration of tools approaching antibiotic efflux that complement routine resistome predictors for preventing antimicrobial therapy failure against difficult-to-threat bacteria.This research was supported by grants MPY 380/18 and MPY 509/19 from the Instituto de Salud Carlos III (ISCIII). ML-S is the recipient of a Sara Borrell contract by the ISCIII. AM-G is the recipient of a Miguel Servet contract by the ISCIII.S

Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae

Streptococcus pneumoniae is the main etiological agent of community-acquired pneumonia and a major cause of mortality and morbidity among children and the elderly. Genome sequencing of several pneumococcal strains revealed valuable information about the potential proteins and genetic diversity of this prevalent human pathogen. However, little is known about its transcriptional regulation and its small regulatory noncoding RNAs. In this study, we performed deep sequencing of the S. pneumoniae TIGR4 strain RNome to identify small regulatory RNA candidates expressed in this pathogen. We discovered 1047 potential small RNAs including intragenic, 5'- and/or 3'-overlapping RNAs and 88 small RNAs encoded in intergenic regions. With this approach, we recovered many of the previously identified intergenic small RNAs and identified 68 novel candidates, most of which are conserved in both sequence and genomic context in other S. pneumoniae strains. We confirmed the independent expression of 17 intergenic small RNAs and predicted putative mRNA targets for six of them using bioinformatics tools. Preliminary results suggest that one of these six is a key player in the regulation of competence development. This study is the biggest catalog of small noncoding RNAs reported to date in S. pneumoniae and provides a highly complete view of the small RNA network in this pathogen.This work was supported by the Fondo de Investigación Sanitaria (FIS) (PI08/0442 and PI11/00656) and the Ministerio de Ciencia e Innovación (MICINN) (Ramón y Cajal program, RYC-2007-00179). A.J.M. was the recipient of a postdoctoral Miguel Servet contract from the Sistema Nacional de Salud funded by the FIS. During the first part of this work, M.A. was the recipient of a junior researcher contract funded by the Ramon y Cajal program from MICINN. S.N. was the recipient of a grant from CIBER Enfermedades Respiratorias (an initiative of the Instituto de Salud Carlos III).S

Predicted Epitope Abundance Supports Vaccine-Induced Cytotoxic Protection Against SARS-CoV-2 Variants of Concern

The effect of emerging SARS-CoV-2 variants on vaccine efficacy is of critical importance. In this study, the potential impact of mutations that facilitate escape from the cytotoxic cellular immune response in these new virus variants for the 551 most abundant HLA class I alleles was analyzed. Computational prediction showed that most of these alleles, that cover >90% of the population, contain enough epitopes without escape mutations in the principal SARS-CoV-2 variants. These data suggest that the cytotoxic cellular immune protection elicited by vaccination is not greatly affected by emerging SARS-CoV-2 variants.This research was supported by grants from COV20_00679 (MPY 222-20), to MM, MPY 509/19 to AM-G, and MPY 388/18 to DL of “Acción Estratégica en Salud” from the ISCIII.S

Bridging Chromosomal Architecture and Pathophysiology of Streptococcus pneumoniae

The chromosome of Streptococcus pneumoniae is organized into topological domains based on its transcriptional response to DNA relaxation: Up-regulated (UP), down-regulated (DOWN), nonregulated (NR), and AT-rich. In the present work, NR genes found to have highly conserved chromosomal locations (17% of the genome) were categorized as members of position-conserved nonregulated (pcNR) domains, while NR genes with a variable position (36% of the genome) were classified as members of position-variable nonregulated (pvNR) domains. On average, pcNR domains showed high transcription rates, optimized codon usage, and were found to contain only a small number of RUP/BOX/SPLICE repeats. They were also poor in exogenous genes but enriched in leading strand genes that code for proteins involved in primary metabolism with central roles within the interactome. In contrast, pvNR genes coding for cell wall proteins, paralogs, virulence factors and immunogenic candidates for protein-based vaccines were found to be overrepresented. DOWN domains were enriched in genes essential for infection. Many UP and DOWN domain genes were seen to be activated during different stages of competence, whereas pcNR genes tended to be repressed until the competence was switched off. Pneumococcal genes appear to be subject to a topology-driven selection pressure that defines the chromosomal location of genes involved in metabolism, virulence and competence. The pcNR domains are interleaved between UP and DOWN domains according to a pattern that suggests the existence of macrodomain entities. The term "topogenomics" is here proposed to describe the study of the topological rules of genomes and their relationship with physiology.This work was supportedby Ministry of Economy and Competitiveness [BIO2014-55462-R]S

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.This research was supported by Acción Estratégica en Salud from the ISCIII (https://www.isciii.es), grants MPY 380/18 (to MJM), 388/18 (to DL) and 509/19 (to AJM-G). AJM-G is the recipient of a Miguel Servet contract by the ISCIII. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Inspecting the potential physiological and biomedical value of 44 conserved uncharacterised proteins of Streptococcus pneumoniae

BACKGROUND: The major Gram-positive coccoid pathogens cause similar invasive diseases and show high rates of antimicrobial resistance. Uncharacterised proteins shared by these organisms may be involved in virulence or be targets for antimicrobial therapy. RESULTS: Forty four uncharacterised proteins from Streptococcus pneumoniae with homologues in Enterococcus faecalis and/or Staphylococcus aureus were selected for analysis. These proteins showed differences in terms of sequence conservation and number of interacting partners. Twenty eight of these proteins were monodomain proteins and 16 were modular, involving domain combinations and, in many cases, predicted unstructured regions. The genes coding for four of these 44 proteins were essential. Genomic and structural studies showed one of the four essential genes to code for a promising antibacterial target. The strongest impact of gene removal was on monodomain proteins showing high sequence conservation and/or interactions with many other proteins. Eleven out of 40 knockouts (one for each gene) showed growth delay and 10 knockouts presented a chaining phenotype. Five of these chaining mutants showed a lack of putative DNA-binding proteins. This suggest this phenotype results from a loss of overall transcription regulation. Five knockouts showed defective autolysis in response to penicillin and vancomycin, and attenuated virulence in an animal model of sepsis. CONCLUSIONS: Uncharacterised proteins make up a reservoir of polypeptides of different physiological importance and biomedical potential. A promising antibacterial target was identified. Five of the 44 examined proteins seemed to be virulence factors.This work was supported by a Miguel Servet Research contract funded by the Fondo de Investigación Sanitaria (Ministerio de Economía y Competitividad de España) to Antonio J. Martin-Galiano, a Plan Nacional de I + D + I of Ministerio de Ciencia e Innovación grant (BIO2011-25343) to Adela G. de la Campa, and funds from the CIBER Enfermedades Respiratorias group (an initiative of the Instituto de Salud Carlos III).S