Genetic reductionist approach for studing the two-component signaling system in Staphylococcus aureus

Staphylococcus aureus es una bacteria ubicua capaz de colonizar una gran variedad de ambientes. En el hombre, S. aureus coloniza las fosas nasales, piel de las axilas, ingles, garganta o incluso el tracto intestinal. Se calcula que un 20% de las personas adultas son portadores nasales de S. aureus. En determinadas circunstancias, la bacteria es capaz de atravesar la barrera epitelial y alcanzar los órganos internos. Cuando esto ocurre, S. aureus se convierte en un patógeno muy versátil capaz de causar enfermedades muy diversas, que pueden ir desde infecciones leves como forúnculos o abscesos hasta enfermedades graves como endocarditis, osteomielitis, neumonía o síndrome del shock tóxico. El desarrollo de S. aureus en distintos ambientes requiere que la bacteria sea capaz de sensar las condiciones ambientales, transmitir los estímulos al citoplasma y activar los cambios necesarios para adecuar la fisiología a dicho ambiente. El principal mecanismo para sensar y responder a las señales ambientales en bacterias son los sistemas de dos-componentes (TCSs). Los TCSs están formados por un sensor de membrana o histidinekinase (HK) y un regulador de respuesta citoplásmico (RR). En el proceso de activación, el sensor recibe su señal específica y se auto fosforila en un dominio histidina. A continuación el fosfato es transferido al residuo aspártico del RR que se encuentra en el citoplasma. De esta forma, el RR se activa y desencadena una respuesta que será acorde a la señal recibida. Normalmente, una bacteria posee varios TCSs, siendo su número proporcional al tamaño del genoma, al número de ambientes distintos en las que es capaz de crecer y a la complejidad de su diferenciación celular. Así, bacterias que viven en ambientes muy constantes, como las bacterias intracelulares estrictas carecen de TCSs, mientras que bacterias que viven en ambientes diversos pueden poseer cientos de ellos. En relación con el número y la función de los TCSs existen varias preguntas que hasta ahora no han sido analizadas: ¿cuántos TCSs necesita una bacteria de vida libre? ¿Son necesarios los TCSs cuando la bacteria crece en un ambiente constante? ¿Existe activación cruzada entre TCSs distintos in vivo? Para responder a estas preguntas y realizar un estudio global de los procesos celulares controlados por los TCSs, en esta tesis hemos realizado una aproximación genética reduccionista usando como modelo dos cepas genéticamente no relacionadas de S. aureus. El trabajo ha consistido en la deleción completa de los 15 TCSs no esenciales que posee S. aureus y la mutación del sensor (WalK) del TCS walKR, cuya deleción completa resulta letal. Las bacterias resultantes carecen del sistema sensorial y su obtención demuestra que en condiciones ambientales constantes estos sistemas son dispensables para la vida de S. aureus. Los mutantes deficientes en los TCSs muestran niveles de crecimiento indistinguibles a los de la cepa salvaje a 37ºC y 44ºC y un patrón metabólico similar. En cambio, los mutantes tienen deficiencias en el crecimiento a 28ºC, pierden la capacidad de reducción de nitratos, muestran mayor sensibilidad al Tritón X-100 así como una menor capacidad para sobrevivir en el ambiente e invadir células. Así mismo, los mutantes tienen reducida su virulencia y capacidad de colonizar órganos en un modelo de infección de ratón. Todos los fenotipos del mutante deficiente en los TCSs podían ser restaurados por la expresión ectópica de un único TCS indicando que cada uno de los fenotipos depende de un único TCS. Finalmente, la cepa deficiente en los TCSs ha sido utilizada como una plataforma para el estudio de la especificidad de transmisión de señal in vivo, un concepto que en inglés se denomina ‘cross-talk’ y que hasta ahora había sido estudiada in vitro. Para ello, hemos establecido una sencilla metodología que consiste en la complementación del mutante deficiente en TCSs con una colección de plásmidos que contienen una combinación de la familia de HKs y un RR. El análisis de las cepas complementadas nos ha permitido identificar la existencia de activación cruzada entre GraS y ArlR. Esta activación cruzada tiene lugar incluso en presencia de sus correspondientes parejas, la HK ArlS y el RR GraR. Teniendo en cuenta que durante este análisis global sólo hemos detectado activación cruzada entre estos TCSs, la conclusión de nuestro estudio es que la activación cruzada entre los TCSs puede ocurrir in vivo, pero no es frecuente. En el futuro las cepas deficientes en los TCSs, o cepas derivadas conteniendo únicamente uno de ellos, servirán para identificar el regulón que controla cada TCS o para identificar nuevos fármacos que bloqueen específicamente a los TCSs.Staphylococcus aureus is a Gram-positive bacterium adapted to live in a wide variety of environmental niches. In humans, S. aureus colonizes the nose, skin, axilla, groin, throat or intestinal tract. Approximately 20% of the anterior nares of healthy human adults are persistently colonized with S. aureus. In some circumstances, the bacterium is able to traverse the epithelial barrier reaching internal organs. When this occurs, S. aureus can cause a variety of diseases, ranging from minor skin infections, such as furuncles or abscesses, to severe infections, such as endocarditis, osteomyelitis, pneumonia or toxic shock syndrome. S. aureus needs to recognize environmental signals, transmit stimuli to the cytoplasm and activate the necessary changes to adapt the bacterial physiology to the conditions of each environmental niche. The main mechanism to sense and respond to environmental signals in bacteria is the two-component transduction system (TCSs). TCSs comprise a membrane sensor histidine kinase (HK) and a cytoplasmic response regulator (RR). During the activation process, the sensor receives a specific signal and autophosphorylates itself on a conserved histidine residue. The phosphate is then transferred to an aspartate residue of the cytoplasmic cognate RR. The phosphorylated RR triggers a specific response in accordance with the signal. The genome of a single bacterial species usually encodes for multiple signal transducers: the number often proportional to the genome size, the diversity of environments in which organisms live and the complexity in cellular differentiation. Bacteria inhabiting relatively stable host environments, such as obligate intracellular parasites, encode for few or even no TCS signalling systems, while ubiquitous bacteria that are able to live in a variety of environments encode high numbers of TCSs. In relation with the number and the function of TCSs in bacteria, several questions remain open: How many TCSs does a free-living bacterium need to live? Are TCSs necessary when bacteria live in a constant environment? Does cross-activation between different TCSs exist in vivo? With the aim to answer these questions and to carry out a global analysis of the cellular processes controlled by TCSs, we generated S. aureus mutants devoid of the TCS signalling networks by using a genetic reductionist approach on two genetically unrelated S. aureus strains. The work consisted in the sequential deletion of the 15 non-essential TCSs of S. aureus as well as the deletion of the sensor (WalK) of the walKR TCS, whose complete deletion is lethal. The resulting mutants lacking the TCSs demonstrate that under constant environmental conditions these systems are dispensable for S. aureus survival. Phenotypic analyses of the mutants devoid of TCSs revealed growth levels indistinguishable from the wild type at 37 and 44ºC, and similar metabolic capacities. However, mutants devoid of TCSs lose the capacity to reduce nitrate to nitrite, show lower growth rates at 28ºC and capacity to survive in the environment and higher sensitivity to detergents. Moreover, in the absence of TCSs, S. aureus is unable to invade eukaryotic cells and colonize organs, rendering the bacteria avirulent in a mouse infection model. Phenotypes associated to the TCS-deficient mutant can be restored by the ectopic expression of single TCSs, indicating that each phenotype is most likely modulated by a single TCS. The TCS-deficient strain was then used as a platform for studying signal transduction specificity (cross-talk) in vivo. For that purpose, we developed a simple method based on the complementation of the TCSdeficient mutant with plasmids that containing a combination of the HK family and a RR. Analysis of these complemented strains allowed the identification of cross-talk between GraS and ArlR. The cross-talk occurs even in the presence of the corresponding ArlS HK and GraR RR. Taking into account that our systematic analysis only found cross-activation between these two TCSs, we conclude that cross-activation between TCSs can occur in vivo, but it is rare. We anticipate that the strains lacking the TCSs, or the set of strains containing single TCSs, will be extremely useful to identify the regulon of each TCS or for finding antimicrobials that specifically block TCS functions.Ministerio de Ciencia e Innovación BIO2008-05284-C02-01; ERA-NET PATHOGENOMICS PIM2010EPI-00606; Ministerio de Ciencia e Innovación BIO2011-30503-C02-02; Ministerio de Economía y Competitividad (BFU2011-23222).Programa Oficial de Doctorado en Biotecnología (RD 1393/2007)Bioteknologiako Doktoretza Programa Ofiziala (ED 1393/2007

Overlapping transcription and bacterial RNA removal

The precise understanding of the biology of a living cell requires the identification and quantification of the molecular components necessary to sustain life. One such element is RNA. Two independent high-throughput strategies are available to identify the entire collection of RNA molecules produced by a cell population, which is currently known as the transcriptome. One technique relies on microarray technology (tiling arrays), whereas the second one relies on sequencing the RNA pool (RNA-seq) (1). Both techniques offer the advantage that the identification of the RNA content is not biased by protein-based genome annotation. The application of these methods to the transcriptome analysis in bacteria has uncovered the existence of a large amount of RNA molecules that overlap at least in some portion with protein-encoding RNA transcripts, generating perfect sense/antisense RNA duplexes (2⇓⇓–5). However, because transcriptome studies have been performed using microgram amounts of RNA purified from millions of bacterial cells instead of RNA purified from a single bacterium, the presence of overlapping sense/antisense RNAs from a genomic region does not necessarily mean that both sense and antisense transcripts are simultaneously present in the same bacteria. Hence, it might be possible that a subgroup in the bacterial population synthesized the sense transcript, another subgroup synthesized the antisense transcript, and consequently overlapping transcripts would never be together in the same cell. A report in PNAS by Lybecker et al. (6) provides clear evidences that both sense and antisense transcripts can be present simultaneously within the same bacterial cell. Using a monoclonal antibody that recognizes double-stranded RNA molecules (dsRNA) irrespectively of the nucleotide sequence, the authors perform immunoprecipitation assays to pull down dsRNA molecules (IP-dsRNA) from a total RNA sample extracted from Escherichia coli, and identified the purified dsRNA by RNA-seq

The role of ArlRS and VraSR in regulating ceftaroline hypersusceptibility in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus infections are a global health problem. New control strategies, including fifth-generation cephalosporins such as ceftaroline, have been developed, however rare sporadic resistance has been reported. Our study aimed to determine whether disruption of two-component environmental signal systems detectably led to enhanced susceptibility to ceftaroline in S. aureus CA-MRSA strain MW2 at sub-MIC concentrations where cells normally continue to grow. A collection of sequential mutants in all fifteen S. aureus non-essential two-component systems (TCS) was first screened for ceftaroline sub-MIC susceptibility, using the spot population analysis profile method. We discovered a role for both ArlRS and VraSR TCS as determinants responsible for MW2 survival in the presence of sub-MIC ceftaroline. Subsequent analysis showed that dual disruption of both arlRS and vraSR resulted in a very strong ceftaroline hypersensitivity phenotype. Genetic complementation analysis confirmed these results and further revealed that arlRS and vraSR likely regulate some common pathway(s) yet to be determined. Our study shows that S. aureus uses particular TCS environmental sensing systems for this type of defense and illustrates the proof of principle that if these TCS were inhibited, the efficacy of certain antibiotics might be considerably enhanced.This work was supported by the Swiss National Science Foundation grants (AR 310030-169404), (WLK 10030-146540 and 10030-192784). MV was supported by FNS (Fonds National Suisse) through project funding 10030-146540. The funders had no role in study design, data collection, and interpretation or the decision to submit the work for publication

A strain of Bacillus thuringiensis containing a novel cry7Aa2 gene that is toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 mu g/mL, which was statistically similar to the estimated LC50 of 20.8 mu g/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 mu g/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.This research was funded by the Programa Nacional de España (No. AGL2015-70584-C2-2-R) and the Gobierno de Navarra (No. IIQ14065: RI1)

Regulation of heterogenous lexA expression in staphylococcus aureus by an antisense RNA originating from transcriptional read-through upon natural mispairings in the sbrB intrinsic terminator

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant no. 646869 to A.T.-A.) and by the Spanish Ministry of Science and Innovation grants (BIO2017-83035-R to I.L. and PID2019-105216GB-I00 to A.T.-A.). Funding for open access charge was provided by the CSIC Open Access Publication Support Initiative, Unit of Information Resources for Research (URICI)

Sensory deprivation in Staphylococcus aureus

Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches.This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, BIO2014-53530-R, SAF2014-56716-REDT, and RTC-2015-3184-1. J.V. was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness