An Uncharacterized Member of the Gls24 Protein Superfamily Is a Putative Sensor of Essential Amino Acid Availability in Streptococcus pneumoniae.

Proteins belonging to the Gls24 superfamily are involved in survival of pathogenic Gram-positive cocci under oligotrophic conditions and other types of stress, by a still unknown molecular mechanism. In Firmicutes, this superfamily includes three different valine-rich orthologal families (Gls24A, B, C) with different potential interactive partners. Whereas the Streptococcus pneumoniae Δgls24A deletion mutant experienced a general long growth delay, the Δgls24B mutant grew as the parental strain in the semisynthetic AGCH medium but failed to grow in the complex Todd-Hewitt medium. Bovine seroalbumin (BSA) was the component responsible for this phenotype. The effect of BSA on growth was concentration-dependent and was maintained when the protein was proteolyzed but not when heat-denatured, suggesting that BSA dependence was related to oligopeptide supplementation. Global transcriptional analyses of the knockout mutant revealed catabolic derepression and induction of chaperone and oligopeptide transport genes. This mutant also showed increased sensibility to cadmium and high temperature. The Δgls24B mutant behaved as a poor colonizer in the nasopharynx of mice and showed 20-fold competence impairment. Experimental data suggest that Gls24B plays a central role as a sensor of amino acid availability and its connection to sugar catabolism. This metabolic rewiring can be compensated in vitro, at the expenses of external oligopeptide supplementation, but reduce important bacteria skills prior to efficiently address systemic virulence traits. This is an example of how metabolic factors conserved in enterococci, streptococci, and staphylococci can be essential for survival in poor oligopeptide environments prior to infection progression.

Boldine-Derived Alkaloids Inhibit the Activity of DNA Topoisomerase I and Growth of Mycobacterium tuberculosis.

The spread of multidrug-resistant isolates of Mycobacterium tuberculosis requires the discovery of new drugs directed to new targets. In this study, we investigated the activity of two boldine-derived alkaloids, seconeolitsine (SCN) and N-methyl-seconeolitsine (N-SCN), against M. tuberculosis. These compounds have been shown to target DNA topoisomerase I enzyme and inhibit growth of Streptococcus pneumoniae. Both SCN and N-SCN inhibited M. tuberculosis growth at 1.95-15.6 µM, depending on the strain. In M. smegmatis this inhibitory effect correlated with the amount of topoisomerase I in the cell, hence demonstrating that this enzyme is the target for these alkaloids in mycobacteria. The gene coding for topoisomerase I of strain H37Rv (MtbTopoI) was cloned into pQE1 plasmid of Escherichia coli. MtbTopoI was overexpressed with an N-terminal 6-His-tag and purified by affinity chromatography. In vitro inhibition of MtbTopoI activity by SCN and N-SCN was tested using a plasmid relaxation assay. Both SCN and N-SCN inhibited 50% of the enzymatic activity at 5.6 and 8.4 µM, respectively. Cleavage of single-stranded DNA was also inhibited with SCN. The effects on DNA supercoiling were also evaluated in vivo in plasmid-containing cultures of M. tuberculosis. Plasmid supercoiling densities were -0.060 in cells untreated or treated with boldine, and -0.072 in 1 × MIC N-SCN treated cells, respectively, indicating that the plasmid became hypernegatively supercoiled in the presence of N-SCN. Altogether, these results demonstrate that the M. tuberculosis topoisomerase I enzyme is an attractive drug target, and that SCN and N-SCN are promising lead compounds for drug development.

Upregulation of the PatAB Transporter Confers Fluoroquinolone Resistance to Streptococcus pseudopneumoniae.

We characterized the mechanism of fluoroquinolone-resistance in two isolates of Streptococcus pseudopneumoniae having fluoroquinolone-efflux as unique mechanism of resistance. Whole genome sequencing and genetic transformation experiments were performed together with phenotypic determinations of the efflux mechanism. The PatAB pump was identified as responsible for efflux of ciprofloxacin (MIC of 4 µg/ml), ethidium bromide (MICs of 8-16 µg/ml) and acriflavine (MICs of 4-8 µg/ml) in both isolates. These MICs were at least 8-fold lower in the presence of the efflux inhibitor reserpine. Complete genome sequencing indicated that the sequence located between the promoter of the patAB operon and the initiation codon of patA, which putatively forms an RNA stem-loop structure, may be responsible for the efflux phenotype. RT-qPCR determinations performed on RNAs of cultures treated or not treated with subinhibitory ciprofloxacin concentrations were performed. While no significant changes were observed in wild-type Streptococcus pneumoniae R6 strain, increases in transcription were detected in the ciprofloxacin-efflux transformants obtained with DNA from efflux-positive isolates, in the ranges of 1.4 to 3.4-fold (patA) and 2.1 to 2.9-fold (patB). Ciprofloxacin-induction was related with a lower predicted free energy for the stem-loop structure in the RNA of S. pseudopneumoniae isolates (-13.81 and -8.58) than for R6 (-15.32 kcal/mol), which may ease transcription. The presence of these regulatory variations in commensal S. pseudopneumoniae isolates, and the possibility of its transfer to Streptococcus pneumoniae by genetic transformation, could increase fluoroquinolone resistance in this important pathogen.

The Transcriptome of Streptococcus pneumoniae Induced by Local and Global Changes in Supercoiling.

The bacterial chromosome is compacted in a manner optimal for DNA transactions to occur. The degree of compaction results from the level of DNA-supercoiling and the presence of nucleoid-binding proteins. DNA-supercoiling is homeostatically maintained by the opposing activities of relaxing DNA topoisomerases and negative supercoil-inducing DNA gyrase. DNA-supercoiling acts as a general cis regulator of transcription, which can be superimposed upon other types of more specific trans regulatory mechanism. Transcriptomic studies on the human pathogen Streptococcus pneumoniae, which has a relatively small genome (∼2 Mb) and few nucleoid-binding proteins, have been performed under conditions of local and global changes in supercoiling. The response to local changes induced by fluoroquinolone antibiotics, which target DNA gyrase subunit A and/or topoisomerase IV, involves an increase in oxygen radicals which reduces cell viability, while the induction of global supercoiling changes by novobiocin (a DNA gyrase subunit B inhibitor), or by seconeolitsine (a topoisomerase I inhibitor), has revealed the existence of topological domains that specifically respond to such changes. The control of DNA-supercoiling in S. pneumoniae occurs mainly via the regulation of topoisomerase gene transcription: relaxation triggers the up-regulation of gyrase and the down-regulation of topoisomerases I and IV, while hypernegative supercoiling down-regulates the expression of topoisomerase I. Relaxation affects 13% of the genome, with the majority of the genes affected located in 15 domains. Hypernegative supercoiling affects 10% of the genome, with one quarter of the genes affected located in 12 domains. However, all the above domains overlap, suggesting that the chromosome is organized into topological domains with fixed locations. Based on its response to relaxation, the pneumococcal chromosome can be said to be organized into five types of domain: up-regulated, down-regulated, position-conserved non-regulated, position-variable non-regulated, and AT-rich. The AT content is higher in the up-regulated than in the down-regulated domains. Genes within the different domains share structural and functional characteristics. It would seem that a topology-driven selection pressure has defined the chromosomal location of the metabolism, virulence and competence genes, which suggests the existence of topological rules that aim to improve bacterial fitness.

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.

Absence of tmRNA Has a Protective Effect against Fluoroquinolones in Streptococcus pneumoniae.

The transfer messenger RNA (tmRNA), encoded by the ssrA gene, is a small non-coding RNA involved in trans-translation that contributes to the recycling of ribosomes stalled on aberrant mRNAs. In most bacteria, its inactivation has been related to a decreased ability to respond to and recover from a variety of stress conditions. In this report, we investigated the role of tmRNA in stress adaptation in the human pathogen Streptococcus pneumoniae. We constructed a tmRNA deletion mutant and analyzed its response to several lethal stresses. The ΔssrA strain grew slower than the wild type, indicating that, although not essential, tmRNA is important for normal pneumococcal growth. Moreover, deletion of tmRNA increased susceptibility to UV irradiation, to exogenous hydrogen peroxide and to antibiotics that inhibit protein synthesis and transcription. However, the ΔssrA strain was more resistant to fluoroquinolones, showing twofold higher MIC values and up to 1000-fold higher survival rates than the wild type. Deletion of SmpB, the other partner in trans-translation, also reduced survival to levofloxacin in a similar extent. Accumulation of intracellular reactive oxygen species associated to moxifloxacin and levofloxacin treatment was also highly reduced (∼100-fold). Nevertheless, the ΔssrA strain showed higher intracellular accumulation of ethidium bromide and levofloxacin than the wild type, suggesting that tmRNA deficiency protects pneumococcal cells from fluoroquinolone-mediated killing. In fact, analysis of chromosome integrity revealed that deletion of tmRNA prevented the fragmentation of the chromosome associated to levofloxacin treatment. Moreover, such protective effect appears to relay mainly on inhibition of protein synthesis, since a similar effect was observed with antibiotics that inhibit that process. The emergence and spread of drug-resistant pneumococci is a matter of concern and these results contribute to a better comprehension of the mechanisms underlying fluoroquinolones action.

Epidemiological and molecular aspects of rifampicin-resistant Staphylococcus aureus isolated from wounds, blood and respiratory samples.

OBJECTIVES: To study the incidence of rifampicin-resistant Staphylococcus aureus in Gipuzkoa, Northern Spain, and to characterize representative resistant isolates and mutations associated with resistance. METHODS: For rifampicin-resistant isolates, the rpoB gene fragment that includes the most frequent mutations conferring rifampicin resistance in S. aureus was amplified and sequenced. The role of new mutations responsible for rifampicin resistance was confirmed by cloning and complementation in trans. Resistant isolates were characterized by multilocus sequence typing and PFGE. RESULTS: Between 1999 and 2008, 0.59% (96/16 348) of S. aureus clinical isolates studied showed rifampicin resistance. Rifampicin resistance was higher in methicillin-resistant S. aureus (MRSA) than in methicillin-susceptible S. aureus (MSSA) (3.26% versus 0.26%; P < 0.001). Twenty-two randomly selected rifampicin-resistant isolates were studied in depth, 11 showing low-level and 11 showing high-level rifampicin resistance (rifampicin MICs of 2-4 mg/L and ≥8 mg/L, respectively). Overall, 12 different mutations in the rpoB gene were detected, including a newly described N474K mutation followed by the insertion of a glycine residue at position 475. Among the eight different sequence types (STs) found, the most frequent were ST8 and ST863, the latter being associated with respiratory infections. Ten of the 11 low-level rifampicin-resistant isolates were MRSA ST8 and had the same H481N mutation, while the 11 high-level rifampicin-resistant isolates, 6 MSSA and 5 MRSA, belonged to eight different STs and had distinct rpoB mutations. CONCLUSIONS: Low-level rifampicin-resistant isolates were mainly clonal while high-level resistant isolates showed a high genetic diversity. Most mutations observed coincided with those found in other studies, but a new mutation conferring rifampicin resistance was detected.

Transcriptional analysis of the acid tolerance response in Streptococcus pneumoniae.

Streptococcus pneumoniae, one of the major causes of morbidity and mortality in humans, faces a range of potentially acidic conditions in the middle and late stages of growth in vitro, in diverse human fluids during the infection process, and in biofilms present in the nasopharynx of carriers. S. pneumoniae was shown to develop a weak acid tolerance response (ATR), where cells previously exposed to sublethal pHs (5.8-6.6) showed an increased survival rate of up to one order of magnitude after challenge at the lethal pH (4.4, survival rate of 10(-4)). Moreover, the survival after challenge of stationary phase cells at pH 4.4 was three orders of magnitude higher than that of cells taken from the exponential phase, due to the production of lactic acid during growth and increasing acidification of the growth medium until stationary phase. Global expression analysis after short-term (5, 15 and 30 min, the adaptation phase) and long-term (the maintenance phase) acidic shock (pH 6.0) was performed by microarray experiments, and the results were validated by real-time RT-PCR. Out of a total of 126 genes responding to acidification, 59 and 37 were specific to the adaptation phase and maintenance phase, respectively, and 30 were common to both periods. In the adaptation phase, both up- and down-regulation of gene transcripts was observed (38 and 21 genes, respectively), whereas in the maintenance phase most of the affected genes were down-regulated (34 out of 37). Genes involved in protein fate (including those involved in the protection of the protein native structure) and transport (including transporters of manganese and iron) were overrepresented among the genes affected by acidification, 8.7 and 24.6 % of the acid-responsive genes compared to 2.8 % and 9.6 % of the genome complement, respectively. Cross-regulation with the response to oxidative and osmotic stress was observed. Potential regulatory motifs involved in the ATR were identified in the promoter regions of some of the regulated genes.