BUSCADOR RECOLECTA

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the ter S gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria., Funding for open access charge: Ministerio de Ciencia e Innovación (MICINN) [Consolider-Ingenio CSD2009-00006, BIO2011-30503-C02-01 and Eranet-pathogenomics PIM2010EPA-00606 to J.R.P]; Cardenal Herrera-CEU University [Copernicus-Santander program to J.R.P.]; Insituto Nacional de Investigaciones Agrarias (INIA) [DR08-0093 to M.A.T-M.]; National Institute of Health [R56AI081837 to G.E.C, R01AI022159-23A2 to R.P.N.].

Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through
the consumption of contaminated food, with chicken and pig related products being key reservoirs
of infection. Although numerous studies on animal vaccination have been performed
in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here,
with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly
analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi-GMP
is an intracellular second messenger that controls a wide range of bacterial processes,
including biofilm formation and synthesis of virulence factors, and also modulates
the host innate immune response. Our results showed that a Salmonella multiple mutant in
the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot
synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection
model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that
led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice
against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity
relied on activation of both antibody and cell mediated immune responses characterized by
the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF-
α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation
conditions, indicating that it could be easily eliminated from the environment. Moreover,
ΔXIII shows DIVA features that allow differentiation of infected and vaccinated
animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine, CG and BG are recipients of a
postdoctoral contract under Grants IIM 13329.RI1
and BIO2011-30503-C02-02, respectively. This work
was supported by grant IIM 13329.RI1 from the
Departamento de Innovación, Empresa y Empleo,
Government of Navarra and grants BIO2011-30503-
C02-02 and BIO2014-53530-R from the Spanish
Ministry of Economy and Competitiveness.

Biofilm development and quorum sensing are closely interconnected processes. Biofilm
formation is a cooperative group behaviour that involves bacterial populations living
embedded in a self produced extracellular matrix. Quorum sensing (QS) is a cell-cell
communication mechanism that synchronizes gene expression in response to population
cell density. Intuitively, it would appear that QS might coordinate the switch to a
biofilm lifestyle when the population density reaches a threshold level. However,
compelling evidence obtained in different bacterial species coincides in that activation
of QS occurs in the formed biofilm and activates the maturation and disassembly of the
biofilm in a coordinate manner. The aim of this review is to illustrate, using four
bacterial pathogens as examples, the emergent concept that QS activates the biofilm
dispersion process., Work in the Laboratory of Microbial Biofilms is funded by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, as well as ERA-NET Pathogenomics (PIM2010EPA-00606) and grant from the Departamento de Innovación (IIM13329.RI1), Gobierno de Navarra.

This is an accepted manuscript of an article published by Taylor & Francis in RNA Biology on August 1st 2012, available online: http://dx.doi.org/10.4161/rna.21167., Analysis of bacterial transcriptomes have shown the existence of a genome-wide process of overlapping transcription due to the presence of antisense RNAs, as well as mRNAs that overlapped in their entire length or in some portion of the 5′- and 3′-UTR regions. The biological advantages of such overlapping transcription are unclear but may play important regulatory roles at the level of transcription, RNA stability and translation. In a recent report, the human pathogen Staphylococcus aureus is observed to generate genome-wide overlapping transcription in the same bacterial cells leading to a collection of short RNA fragments generated by the endoribonuclease III, RNase III. This processing appears most prominently in Gram-positive bacteria. The implications of both the use of pervasive overlapping transcription and the processing of these double stranded templates into short RNAs are explored and the consequences discussed., A.T.-A. is recipient of “Ramon y Cajal” contracts from the Spanish Ministry of Science and Innovation. This research was supported by grants ERA-NET Pathogenomics (PIM2010EPA-00606), BIO2008-05284-C02, BIO2011-30503-C02 and BFU2011-23222 from Spanish Ministry of Economy and Competitiveness.

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)

En esta tesis demostramos la existencia de un nivel superior de organización en la estructura del operón que denominamos operón no-contiguo y que consiste en un operón que contiene un gen o genes que se transcriben en la dirección opuesta al resto del operón. Esta nueva arquitectura de transcripción está ejemplificada por los genes menE-menC-MW1733-ytkD-MW1731 que están implicados en la síntesis de menaquinona en Staphylococcus aureus. Nuestros resultados indican que los genes menE-menC-ytkD-MW1731 se transcriben como una unidad de transcripción, mientras el gen MW1733, ubicado entre menC e ytkD, se transcribe en dirección opuesta. Esta organización genética genera transcritos solapantes cuya expresión está regulada a través del procesamiento mediado por RNasa III y por un mecanismo de interferencia transcripcional. El operón menE-menC-MW1733-ytkD-MW1731 es necesario para la síntesis de menaquinona, un intermediario de la cadena transportadora que tiene por función ceder los electrones a los citocromos. La sobreexpresión de MW1733 es capaz de reducir los niveles de menE-menC y en consecuencia la síntesis de menaquinona reduciendo la velocidad de crecimiento de la bacteria y produciendo colonias de tamaño muy pequeño, que se denominan Small Colony Variants (SCV). El fenotipo SCV es característico de aislados clínicos de S. aureus procedentes de infecciones persistentes y es un fenotipo que revierte cuando las bacterias se crecen en el laboratorio. Nuestros resultados indican que la arquitectura de transcripción del operón menE-menC‑MW1733-ytkD-MW1731 podría explicar la variación de fase característico del fenotipo SCV., In this thesis, we report the existence of a higher level of organization in operon structure that we named non‑contiguous operon and consists in an operon containing a gene (s) that is transcribed in the opposite direction to the rest of the operon. This novel transcriptional architecture is exemplified by the genes menE-menC-MW1733-ytkD-MW1731 involved in menaquinone synthesis in the major human pathogen Staphylococcus aureus. We show that menE-menC‑ytkD‑MW1731 genes are transcribed as a single transcription unit whereas the MW1733 gene, located between menC and ytkD, is transcribed in the opposite direction. This genomic organization generates overlapping transcripts whose expression is mutually regulated by transcriptional interference and RNase III processing at the overlapping region.
The menE-menC-MW1733-ytkD-MW1731 operon is necessary for the synthesis of the menaquinone, an intermediary of the electron transport chain. Our results revealed that the overexpression of MW1733 can diminish the expression of MenE-MenC and the menaquinone synthesis and a consequence bacterial growth rate is reduced and form colonies of small size, currently known as Small Colony Variants (SCV). The SCV phenotype is characteristic of clinical isolates of S. aureus obtained from patients with persistent infections. The SCV phenotype is unstable and it reverts when bacteria are grown in the laboratory. Our results suggest that transcriptional organization of the menE-menC‑MW1733-ytkD-MW1731 non-contiguous operon may explain the phenotypic variation of the SCV phenotype., Este trabajo ha sido realizado dentro de los siguientes proyectos de investigación: ‘Estudio de la transferencia horizontal de genes y de la formación del biofilm a nivel de célula individual en Staphylococcus aureus’. BIO2011-30503-C02-02 MINECO. ‘Descifrando las singularidades del exopolisacárido universal del biofilm (PNAG) y evaluación de su potencial biotecnológico’. BIO2014-53530‑R MINECO., Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Recent insights into bacterial biofilm matrix structures have induced a paradigm shift toward the recognition of amyloid fibers
as common building block structures that confer stability to the exopolysaccharide matrix. Here we describe the functional amyloid
systems related to biofilm matrix formation in both Gram-negative and Gram-positive bacteria and recent knowledge regarding
the interaction of amyloids with other biofilm matrix components such as extracellular DNA (eDNA) and the host immune
system. In addition, we summarize the efforts to identify compounds that target amyloid fibers for therapeutic purposes
and recent developments that take advantage of the amyloid structure to engineer amyloid fibers of bacterial biofilm matrices for
biotechnological applications., This work, including the efforts of Jaione Valle, was funded by Ministerio
de Economía y Competitividad (MINECO) (AGL2011-23954). This
work, including the efforts of Íñigo Lasa, was funded by Ministerio de
Economía y Competitividad (MINECO) (BIO2011-30503-C02-02 and
BIO2014-53530-R).

Esta es la versión no revisada del artículo: Valle, J., Burgui, S.,
Langheinrich, D., Gil, C., Solano, C., Toledo-Arana, A., et al. (2015). Evaluation
of Surface Microtopography Engineered by Direct Laser Interference for Bacterial
Anti-Biofouling. Macromolecular Bioscience, 15(8), 1060–1069. Se puede consultar la versión publicada en http://doi.org/10.1002/mabi.201500107, Biofilm formation by bacterial pathogens on the surface of medical and industrial settings is a 25 serious health problem. Modification of the biomaterial surface topography is a promising 26 strategy to prevent bacterial attachment and biofilm development. However, fabrication of 27 functional biomaterials at large scale with periodic network-topology is still problematic. In this 28 study, we use direct laser interference (DLIP), an easily scalable process, to modify polystyrene 29 surface (PS) topography at sub-micrometer scale. The resulting structure surfaces were 30 interrogated for their capacity to prevent adhesion and biofilm formation of the major human 31 pathogen Staphylococcus aureus. The results revealed that three-dimensional micrometer 32 periodic structures on PS have a profound impact on bacterial adhesion capacity. Thus, line- 33 and pillar-like topographical patterns enhanced S. aureus adhesion, whereas complex lamella 34 microtopography reduced S. aureus adhesion both in static and continuous flow culture 35 conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus 36 adhesion both when the surface is coated with human serum proteins in vitro and when the 37 material is implanted subcutaneously in a foreign-body associated infection model. Our results 38 establish that the DLIP technology can be used to functionalize polymeric surfaces for the 39 inhibition of bacterial adhesion to surfaces., J. Valle was supported by Spanish Ministry of Science and Innovation “Ramón y Cajal” 369 contract. This research was supported by grants AGL2011-23954 and BIO2011-30503-C02-02 370 from the Spanish Ministry of Economy and Competitivity and IIQ14066.RI1 from Innovation 371 Department of the Government of Navarra.

Incluye dos ficheros de datos, The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due
to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the
formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated
infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we
analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S.
aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased
biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature
of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration
of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the
production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and
killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside
a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized
mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle.
Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine
against S. aureus biofilm-associated infections., J. Valle was supported by Spanish Ministry of Science and Innovation
“Ramón y Cajal” contract. This research was supported by grants ERANET
Pathogenomic (GEN2006-27792-C2-1-E/PAT), BIO2011-30503-
C02-02, and AGL2011-23954 from the Spanish Ministry of Economy and
Competitivity and IIQ14066.RI1 from Innovation Department of the
Government of Navarra.

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.

Biofilm development is recognized as a major virulence factor underlying most chronic bacterial infections. When a biofilm community is established, planktonic cells growing in the surroundings of a tissue switch to a sessile lifestyle and start producing a biofilm matrix. The initial steps of in vivo biofilm development are poorly characterized and difficult to assess experimentally. A great amount of in vitro evidence has shown that accumulation of high levels of cyclic dinucleotides (c-di-NMPs) is the most prevalent hallmark governing the initiation of biofilm development by bacteria. As mentioned above, recent studies also link detection of c-di-NMPs by host cells with the activation of a type I interferon immune response against bacterial infections. We discuss here c-di-NMP signaling and the host immune response in the context of the initial steps of in vivo biofilm development., J. Valle was supported by Spanish Ministry of Science and Innovation ‘Ramón y Cajal’ contract. Work in the Laboratory of Microbial Biofilms is funded by the Spanish Ministry of Economy and Competitiveness grants BIO2011-30503-C02-02, AGL2011-23954, and BFU2011-23222, as well as by ERA-NET Pathogenomics (PIM2010EPA-00606) and grants from the Departamento de Innovación (IIQ14066.RI1 and IIM13329.RI1) and Departamento de Salud (Resolución 1312/2010), Gobierno de Navarra.