BUSCADOR RECOLECTA

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.

Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization., JV was supported by SAF2015-74267-JIN. This research was supported by grants BIO2014-53530-R, BIO2017-83035-R and RTC-2015-3184-1 from the Spanish Ministry of Economy and Competitivity.

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.

Incluye material complementario, Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di-GMP (c-di-GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities.

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or
PNAG (poly-β-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members
of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis,
Salmonella lacks it. The evolutionary force driving this difference remains to be determined.
Here, we report that Salmonella lost the pgaABCD operon after the divergence
of Salmonella and Citrobacter clades, and previous to the diversification of the currently
sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella
with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner.
Outside the host, the PGA polysaccharide does not seem to provide any significant benefit
to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal
stress and phage infection remained the same as in a strain producing cellulose, the main
biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production
proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility
to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive
inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether,
our observations indicate that PGA is an antivirulence factor whose loss may have
been a necessary event during Salmonella speciation to permit survival inside the host., This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2014-53530-R and SAF2014-56716-REDT. JV was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness.

RNA-binding proteins (RBPs) are essential to finetune
gene expression. RBPs containing the coldshock
domain are RNA chaperones that have been
extensively studied. However, the RNA targets and
specific functions for many of them remain elusive.
Here, combining comparative proteomics and RBPimmunoprecipitation-
microarray profiling, we have
determined the regulon of the RNA chaperone CspA
of Staphylococcus aureus. Functional analysis revealed
that proteins involved in carbohydrate and ribonucleotide
metabolism, stress response and virulence
gene expression were affected by cspA
deletion. Stress-associated phenotypes such as increased
bacterial aggregation and diminished resistance
to oxidative-stress stood out. Integration of
the proteome and targetome showed that CspA posttranscriptionally
modulates both positively and negatively
the expression of its targets, denoting additional
functions to the previously proposed translation
enhancement. One of these repressed targets
was its own mRNA, indicating the presence of a
negative post-transcriptional feedback loop. CspA
bound the 5 UTR of its own mRNA disrupting a hairpin,
which was previously described as an RNase III
target. Thus, deletion of the cspA 5 UTR abrogated
mRNA processing and auto-regulation. We propose
that CspA interacts through a U-rich motif, which
is located at the RNase III cleavage site, portraying
CspA as a putative RNase III-antagonist., European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme [646869]; Spanish Ministry of Economy and Competitiveness [BFU2011-23222, BIO2014-53530-R, BFU2014-56698-P]; Spanish National Research Council [CSIC-PII-201540I013]; C.J.C. was supported by predoctoral contract from the Public University of Navarre (UPNA), Spain. Funding for open access charge: European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme [646869].

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer, Parkinson or Type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity towards that diversity of amyloid fibrils, or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here, we show the feasibility of the approach by designing, synthesizing and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The more soluble derivative, compound 1D, stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein or amylin, provides a sensitivity only slightly lower than that of Thioflavin T, displays a large Stokes shift, allows an efficient excitation in the UV spectral region,and it is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by Staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining,S. aureus biofilms containing amyloid forming phenol soluble modulins from those lacking them., IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SVis supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government.

Despite the number of examples that correlate interspecies interactions in polymicrobial infections with variations in pathogenicity and antibiotic susceptibility of individual organisms, antibiotic therapies are selected to target the most relevant pathogen, with no consideration of the consequences that the presence of other bacterial species may have in the pathogenicity and response to antimicrobial agents. In this issue of Virulence, Garcia-Perez et al. [ 10 ] applied replica plating of used wound dressings to assess the topography of distinct S. aureus types in chronic wounds of patients with the genetic blistering disease epidermolysis bullosa, which is characterized by the development of chronic wounds upon simple mechanical trauma. This approach led to the identification of two strains of S. aureus coexisting with Bacillus thuringiensis and Klebsiella oxytoca. S. aureus is highly prevalent in chronic wound infections, whereas B. thuringiensis and K. oxytoca are regarded as opportunistic pathogens. These bacterial species did not inhibit each other's growth under laboratory conditions, suggesting that they do not compete through the production of inhibitory compounds. Using a top-down proteomic approach to explore the inherent relationships between these co-existing bacteria, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis or K. oxytoca were characterized by Mass Spectrometry., Ministerio de Economía y Competitividad (BIO2014-53530-R).

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)

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA)., SB was supported by a predoctoral contract from the Public University of Navarra. CG, ME and BG were recipients of postdoctoral contracts under Grant BIO2014-53530-R. This research was supported by grant IIM 13329.RI1 from the Departamento de Innovación, Empresa y Empleo, Government of Navarra and grants BIO2014-53530-R and BIO2017-83035-R from the Spanish Ministry of Economy and Competitiveness (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union).

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).

En este trabajo de tesis hemos evaluado dos aproximaciones diferentes dedicadas a la búsqueda de fármacos que tengan como diana de acción los TCSs. Por un lado, dentro del proyecto 'Nuevas estrategias para el control de infecciones nosocomiales (RTC-2015-3184-1) hemos participado en la evaluación de una colección de fármacos aprobados por la FDA (colección Prestwick) para distintas patologías como posibles agentes antimicrobianos frente a S. aureus. El objetivo era reconvertir alguno de estos fármacos (repurposing) y que además de utilizarse para la finalidad para la que fue descrito, también actuara como antimicrobiano. En el caso concreto de nuestro estudio, el escrutinio se hizo frente al TCS GraRS. La segunda estrategia, recogida en el cuarto capítulo de esta tesis, ha consistido en el desarrollo de dos terapias alternativas frente a S. aureus basadas en inmunoterapias: anticuerpos monoclonales y nanoanticuerpos. Para ello, hemos elegido como diana de acción la HK del TCS esencial WalKR, en particular los anticuerpos se han generado frente al dominio de unión a ligando (ligand-bindig domain, LBD) de la HK. La elección de esta diana se debe a dos razones, por un lado, se trata del único TCS esencial de S. aureus y, por otro lado, ensayos realizados con sueros de pacientes infectados por S. aureus mostraron la presencia de anticuerpos frente a este dominio. A lo largo de la tesis hemos producido anticuerpos monoclonales y nanoanticuerpos frente a diferentes epítopos del LBD. Hemos realizado diferentes ensayos para probar el reconocimiento de la proteína tanto in vitro como in vivo y para analizar el efecto de los anticuerpos en el crecimiento de S. aureus. Los ensayos preliminares indican que varios de los nanoanticuerpos desarrollados activan el crecimiento de la bacteria en lugar de reducir su crecimiento. Este resultado, aunque inesperado, indica que es posible desarrollar nanoanticuerpos capaces de afectar la funcionalidad de la HK y por tanto de bloquear su actividad., During the course of the thesis we have participated in the development of two potential complementary therapies against S. aureus. The first strategy was carried out within the framework of the project ‘New strategies to control nosocomial infections’ (RTC-2015-3184-1) and we have participated in the screening of a collection of FDA’s approved drugs (Prestwick collection) as new antimicrobial agents. The objective was to repurpose one the compounds as a new drug against S. aureus, in this case the target of the screening was the TCS GraRS. The second strategy is presented in the fourth chapter of the thesis. The goal was to develop a potential complementary therapy against S. aureus based on two different immunetherapy strategies: monoclonal antibodies and nanobodies. For that, we have focused on the HK of S. aureus and in particular the antibodies were developed against the ligand-binding domain (LBD) of the HK from WalRK. We have selected this target because WalRK is the only essential TCS of S. aureus and besides, assays performed with serum from patients with an S. aureus infection showed an immunogenic response against the LBD. We have been able to obtain both, the monoclonal antibody and also different nanobodies against the LBD region of WalK able to recognize the peptide in vitro and in vivo. Unexpectedly, incubation of S. aureus with various nanobodies promoted instead of inhibited bacterial growth. These surprising results indicate that nanobodies can modify the activity of HKs and as a consequence, interfere with the signal transduction system., Este trabajo ha sido realizado dentro de los siguientes proyectos de investigación: ‘Descifrando las singularidades del exopolisacárido universal del biofilm (PNAG) y evaluación de su potencial biotecnológico’. BIO2014-53530-R MINECO. ‘Nuevas estrategias para el control de infecciones nosocomiales’. RTC-2015-3184-1, MINECO. ‘Caracterización funcional de los determinantes moleculares para la adaptación de Staphylococcus aureus a la virulencia’. BIO2017-83035-R. MINECO, Programa de Doctorado en Biotecnología (RD 99/2011), Bioteknologiako Doktoretza Programa (ED 99/2011)

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This 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. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigacion/Fondo Europeo de Desarrollo Regional, European Union). A.T.-A. is supported by the European Research Council under the European Union's Horizon 2020 research and innovation programme Grant Agreement 646869.

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.

Major components of the biofilm matrix scaffold are proteins that assemble to create a unified structure that maintain bacteria attached to each other and to surfaces. We provide evidence that a surface protein present in several staphylococcal species forms functional amyloid aggregates to build the biofilm matrix in response to specific environmental conditions. Under low Ca2+ concentrations and acidic pH, Bap is processed and forms insoluble aggregates with amyloidogenic properties. When the Ca2+ concentration increases, metal-coordinated Bap adopts a structurally more stable conformation and as a consequence, the N-terminal region is unable to assemble into amyloid aggregates. The control of Bap cleavage and assembly helps to regulate biofilm matrix development as a function of environmental changes., This research was supported by the
Spanish Ministry of Economy and Competitiveness
grants AGL2011-23954, BIO2014-53530-R and
BFU2013-44763-P. JV was supported by Ramon y
Cajal (RYC-2009-03948) contract from the Spanish
Ministry of Economy and Competitiveness.

Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids., This work was financially supported by the Spanish Ministry of Science, Innovationand Universities grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S.
P.D.-M. was supported by an F.P.I. (BES-2015-072859) contract from the Spanish Ministry of Science, Innovation and Universities.

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5'UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5'UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.

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 Lasa Iñigo, was funded by Ministerio de Economía y Competitividad (MINECO) (BIO2011-30503-C02-02 and BIO2014-53530-R, Peer Reviewed

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., SB is a predoctoral fellow from the Public University of Navarra. 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., Peer Reviewed

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria., This research was supported by the Spanish Ministry of Economy and Competitiveness grants AGL2011-23954, BIO2014-53530-R and BFU2013-44763-P. JV was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness., Peer Reviewed

12 Páginas, 7 Figuras. Contiene información suplementaria en: http://dx.doi.org/10.1038/s41467-018-02949-y, 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., Peer reviewed

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them., IL is supported by the Spanish Ministry of Economy and Competitiveness grant BIO2014-53530-R. SV is supported by grant BIO2016-783-78310-R and by ICREA (ICREA Academia 2015). MDD is supported by the Government of Aragon (GA E-102). JS is supported by grants BFU2016-78232-P (MINECO, Spain) and E45_17R (Gobierno de Aragón, Spain). JS and IL acknowledge financial support from grant CI-2017/001-3 (Campus Iberus, Spain). AM was a recipient of a predoctoral FPU fellowship from the Spanish Government., Peer reviewed

RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5′UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5′UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist., European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme [646869]; Spanish Ministry of Economy and Competitiveness [BFU2011-23222, BIO2014-53530-R, BFU2014-56698-P]; Spanish National Research Council [CSIC-PII-201540I013]; C.J.C. was supported by predoctoral contract from the Public University of Navarre (UPNA), Spain. Funding for open access charge: European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme [646869]., Peer reviewed

Bacterial genes are typically grouped into operons defined as clusters of adjacent genes encoding for proteins that fill related roles and are transcribed into a single polycistronic mRNA molecule. This simple organization provides an efficient mechanism to coordinate the expression of neighboring genes and is at the basis of gene regulation in bacteria. Here, we report the existence of a higher level of organization in operon structure that we named noncontiguous operon and consists in an operon containing a gene(s) that is transcribed in the opposite direction to the rest of the operon. This 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. In light of our results, the canonical view of operon structure should be revisited by including this operon arrangement in which cotranscription and overlapping transcription are combined to coordinate functionally related gene expression., This work was supported by the Spanish Ministry of Economy and
Competitiveness Grants BIO2014-53530-R and BIO2017-83035-R (Agencia
Española de Investigación/Fondo Europeo de Desarrollo Regional, European
Union). A.T.-A. is supported by the European Research Council under the
European Union’s Horizon 2020 research and innovation programme Grant
Agreement 646869., Peer reviewed

15 p.-4 fig.-2 tab., Bacterial biofilms provide high cell density and a superior adaptation and protection from stress conditions compared to planktonic cultures, making them a very promising approach for bioremediation. Several Rhodococcus strains can desulfurize dibenzothiophene (DBT), a major sulphur pollutant in fuels, reducing air pollution from fuel combustion. Despite multiple efforts to increase Rhodococcus biodesulfurization activity, there is still an urgent need to develop better biocatalysts. Here, we implemented a new approach that consisted in promoting Rhodococcus erythropolis biofilm formation through the heterologous expression of a diguanylate cyclase that led to the synthesis of the biofilm trigger molecule cyclic di‐GMP (c‐di‐GMP). R. erythropolis biofilm cells displayed a significantly increased DBT desulfurization activity when compared to their planktonic counterparts. The improved biocatalyst formed a biofilm both under batch and continuous flow conditions which turns it into a promising candidate for the development of an efficient bioreactor for the removal of sulphur heterocycles present in fossil fuels., This study was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014‐53530‐R and BIO2017‐83035‐R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I. Lasa and C. Solano and grants BIO2016‐79736‐R, PCIN‐2014‐113 and PCI2019‐111833‐2 to E. Díaz. P. Dorado‐Morales was supported by a F.P.I. (BES‐2015‐072859) contract from the Spanish Ministry of Science, Innovation and Universities., Peer reviewed

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-β-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host., [Author summary] During bacterial evolution, specific traits that optimize the organism’s fitness are selected. The production of exopolysaccharides is widespread among bacteria in which they play a protective shielding role as main constituents of biofilms. In contrast to closely related siblings, Salmonella has lost the capacity to produce the exopolysaccharide PGA. Our study reveals that Salmonella lost pga genes, and that the driving force for such a loss may have been the detrimental impact that PGA has during Salmonella invasion of internal organs where it augments the susceptibility to bile salts and oxygen radicals, reducing bacterial survival inside macrophages and rendering Salmonella avirulent. These results suggest that gene-loss has played an important role during Salmonella evolution., This work was supported by the Spanish Ministry of Economy and Competitiveness grants BIO2014-53530-R and SAF2014-56716-REDT (http://www.mineco.gob.es/portal/site/mineco/?lang_choosen=en). JV was supported by Ramon y Cajal (RYC-2009-03948) contract from the Spanish Ministry of Economy and Competitiveness.

Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization., JV was supported by SAF2015-74267-JIN. This research was supported by grants BIO2014-53530-R, BIO2017-83035-R and RTC-2015-3184-1 from the Spanish Ministry of Economy and Competitivity.

© 2021 Dorado-Morales et al., Plasmids have largely contributed to the spread of antimicrobial resistance genes among Staphylococcus strains. Knowledge about the fitness cost that plasmids confer on clinical staphylococcal isolates and the coevolutionary dynamics that drive plasmid maintenance is still scarce. In this study, we aimed to analyze the initial fitness cost of plasmids in the bacterial pathogen Staphylococcus aureus and the plasmid-host adaptations that occur over time. For that, we first designed a CRISPR (clustered regularly interspaced palindromic repeats)-based tool that enables the removal of native S. aureus plasmids and then transferred three different plasmids isolated from clinical S. aureus strains to the same-background clinical cured strain. One of the plasmids, pUR2940, obtained from a livestock-associated methicillin-resistant S. aureus (LA-MRSA) ST398 strain, imposed a significant fitness cost on both its native and the new host. Experimental evolution in a nonselective medium resulted in a high rate pUR2940 loss and selected for clones with an alleviated fitness cost in which compensatory adaptation occurred via deletion of a 12.8-kb plasmid fragment, contained between two ISSau10 insertion sequences and harboring several antimicrobial resistance genes. Overall, our results describe the relevance of plasmid-borne insertion sequences in plasmid rearrangement and maintenance and suggest the potential benefits of reducing the use of antibiotics both in animal and clinical settings for the loss of clinical multidrug resistance plasmids., This work was financially supported by the Spanish Ministry of Science, Innovation and Universities grants BIO2014-53530-R and BIO2017-83035-R (Agencia Española de Investigación/Fondo Europeo de Desarrollo Regional, European Union) to I.L. and C.S. P.D.-M. was supported by an F.P.I. (BES-2015-072859) contract from the Spanish Ministry of Science, Innovation and Universities.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer’s disease, Parkinson’s disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward that diversity of amyloid fibrils or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here we show the feasibility of the approach by designing, synthesizing, and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aß1–42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1–42, a-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them.