Modern Acinetobacter baumannii clinical isolates replicate inside spacious vacuoles and egress from macrophages.

Multidrug-resistant Acinetobacter baumannii infections are increasing at alarming rates. Therefore, novel antibiotic-sparing treatments to combat these A. baumannii infections are urgently needed. The development of these interventions would benefit from a better understanding of this bacterium's pathobiology, which remains poorly understood. A. baumannii is regarded as an extracellular opportunistic pathogen. However, research on Acinetobacter has largely focused on common lab strains, such as ATCC 19606, that have been isolated several decades ago. These strains exhibit reduced virulence when compared to recently isolated clinical strains. In this work, we demonstrate that, unlike ATCC 19606, several modern A. baumannii clinical isolates, including the recent clinical urinary isolate UPAB1, persist and replicate inside macrophages within spacious vacuoles. We show that intracellular replication of UPAB1 is dependent on a functional type I secretion system (T1SS) and pAB5, a large conjugative plasmid that controls the expression of several chromosomally-encoded genes. Finally, we show that UPAB1 escapes from the infected macrophages by a lytic process. To our knowledge, this is the first report of intracellular growth and replication of A. baumannii. We suggest that intracellular replication within macrophages may contribute to evasion of the immune response, dissemination, and antibiotic tolerance of A. baumannii.

Structure of the putative long tail fiber receptor-binding tip of a novel temperate bacteriophage from the Antarctic bacterium Bizionia argentinensis JUB59.

Tailed bacteriophages are one of the most widespread biological entities on Earth. Their singular structures, such as spikes or fibers are of special interest given their potential use in a wide range of biotechnological applications. In particular, the long fibers present at the termini of the T4 phage tail have been studied in detail and are important for host recognition and adsorption. Although significant progress has been made in elucidating structural mechanisms of model phages, the high-resolution structural description of the vast population of marine phages is still unexplored. In this context, we present here the crystal structure of C24, a putative receptor-binding tip-like protein from Bizionia argentinensis JUB59, a psychrotolerant bacterium isolated from the marine surface waters of Potter Cove, Antarctica. The structure resembles the receptor-binding tip from the bacteriophage T4 long tail fiber yet showing marked differences in its domain organization, size, sequence identity and metal binding nature. We confirmed the viral origin of C24 by induction experiments using mitomycin C. Our results reveal the presence of a novel uncharacterized prophage in the genome of B. argentinensis JUB59, whose morphology is compatible with the order Caudovirales and that carries the nucleotide sequence of C24 in its genome. This work provides valuable information to expand our current knowledge on the viral machinery prevalent in the oceans.

ChIP-seq analysis of the LuxR-type regulator VjbR reveals novel insights into the Brucella virulence gene expression network.

LuxR-type transcription factors control diverse physiological functions necessary for bacterial adaptation to environmental changes. In the intracellular pathogen Brucella, the LuxR homolog VjbR has been shown to regulate the expression of virulence factors acting at early stages of the intracellular infection and, directly or indirectly, hundreds of additional genes. However, the precise determination of VjbR direct targets has so far proved elusive. Here, we performed chromatin immunoprecipitation of VjbR followed by next-generation sequencing (ChIP-seq). We detected a large amount of VjbR-binding sites distributed across the Brucella genome and determined a markedly asymmetric binding consensus motif, an unusual feature among LuxR-type regulators. RNA-seq analysis performed under conditions mimicking the eukaryotic intracellular environment revealed that, among all loci associated to VjbR-binding, this regulator directly modulated the expression of only a subset of genes encoding functions consistent with an intracellular adaptation strategy for survival during the initial stages of the host cell infection. Other VjbR-binding events, however, showed to be dissociated from transcription and may require different environmental signals to produce a transcriptional output. Taken together, our results bring new insights into the extent and functionality of LuxR-type-related transcriptional networks.

Xanthomonas campestris attenuates virulence by sensing light through a bacteriophytochrome photoreceptor.

Phytochromes constitute a major photoreceptor family found in plants, algae, fungi, and prokaryotes, including pathogens. Here, we report that Xanthomonas campestris pv. campestris (Xcc), the causal agent of black rot disease which affects cruciferous crops worldwide, codes for a functional bacteriophytochrome (XccBphP). XccBphP possesses an N-terminal PAS2-GAF-PHY photosensory domain triad and a C-terminal PAS9 domain as its output module. Our results show that illumination of Xcc, prior to plant infection, attenuates its virulence in an XccBphP-dependent manner. Moreover, in response to light, XccBphP downregulates xanthan exopolysaccharide production and biofilm formation, two known Xcc virulence factors. Furthermore, the XccbphP null mutant shows enhanced virulence, similar to that of dark-adapted Xcc cultures. Stomatal aperture regulation and callose deposition, both well-established plant defense mechanisms against bacterial pathogens, are overridden by the XccbphP strain. Additionally, an RNA-Seq analysis reveals that far-red light or XccBphP overexpression produces genomewide transcriptional changes, including the inhibition of several Xcc virulence systems. Our findings indicate that Xcc senses light through XccBphP, eliciting bacterial virulence attenuation via downregulation of bacterial virulence factors. The capacity of XccBphP to respond to light both in vitro and in vivo was abolished by a mutation on the conserved Cys13 residue. These results provide evidence for a novel bacteriophytochrome function affecting an infectious process.

A Fijivirus Major Viroplasm Protein Shows RNA-Stimulated ATPase Activity by Adopting Pentameric and Hexameric Assemblies of Dimers.

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.

Dimer Asymmetry and Light Activation Mechanism in Brucella Blue-Light Sensor Histidine Kinase.

The ability to sense and respond to environmental cues is essential for adaptation and survival in living organisms. In bacteria, this process is accomplished by multidomain sensor histidine kinases that undergo autophosphorylation in response to specific stimuli, thereby triggering downstream signaling cascades. However, the molecular mechanism of allosteric activation is not fully understood in these important sensor proteins. Here, we report the full-length crystal structure of a blue light photoreceptor LOV histidine kinase (LOV-HK) involved in light-dependent virulence modulation in the pathogenic bacterium Brucella abortus Joint analyses of dark and light structures determined in different signaling states have shown that LOV-HK transitions from a symmetric dark structure to a highly asymmetric light state. The initial local and subtle structural signal originated in the chromophore-binding LOV domain alters the dimer asymmetry via a coiled-coil rotary switch and helical bending in the helical spine. These amplified structural changes result in enhanced conformational flexibility and large-scale rearrangements that facilitate the phosphoryl transfer reaction in the HK domain.IMPORTANCE Bacteria employ two-component systems (TCSs) to sense and respond to changes in their surroundings. At the core of the TCS signaling pathway is the multidomain sensor histidine kinase, where the enzymatic activity of its output domain is allosterically controlled by the input signal perceived by the sensor domain. Here, we examine the structures and dynamics of a naturally occurring light-sensitive histidine kinase from the pathogen Brucella abortus in both its full-length and its truncated constructs. Direct comparisons between the structures captured in different signaling states have revealed concerted protein motions in an asymmetric dimer framework in response to light. Findings of this work provide mechanistic insights into modular sensory proteins that share a similar modular architecture.

Adhesins of Brucella: Their Roles in the Interaction with the Host.

A central aspect of Brucella pathogenicity is its ability to invade, survive, and replicate in diverse phagocytic and non-phagocytic cell types, leading to chronic infections and chronic inflammatory phenomena. Adhesion to the target cell is a critical first step in the invasion process. Several Brucella adhesins have been shown to mediate adhesion to cells, extracellular matrix components (ECM), or both. These include the sialic acid-binding proteins SP29 and SP41 (binding to erythrocytes and epithelial cells, respectively), the BigA and BigB proteins that contain an Ig-like domain (binding to cell adhesion molecules in epithelial cells), the monomeric autotransporters BmaA, BmaB, and BmaC (binding to ECM components, epithelial cells, osteoblasts, synoviocytes, and trophoblasts), the trimeric autotransporters BtaE and BtaF (binding to ECM components and epithelial cells) and Bp26 (binding to ECM components). An in vivo role has also been shown for the trimeric autotransporters, as deletion mutants display decreased colonization after oral and/or respiratory infection in mice, and it has also been suggested for BigA and BigB. Several adhesins have shown unipolar localization, suggesting that Brucella would express an adhesive pole. Adhesin-based vaccines may be useful to prevent brucellosis, as intranasal immunization in mice with BtaF conferred high levels of protection against oral challenge with B. suis.

MapB, the Brucella suis TamB homologue, is involved in cell envelope biogenesis, cell division and virulence.

Brucella species are Gram-negative, facultative intracellular pathogens responsible for a worldwide zoonosis. The envelope of Brucella exhibits unique characteristics that make these bacteria furtive pathogens and resistant to several host defence compounds. We have identified a Brucella suis gene (mapB) that appeared to be crucial for cell envelope integrity. Indeed, the typical resistance of Brucella to both lysozyme and the cationic lipopeptide polymyxin B was markedly reduced in a ∆mapB mutant. MapB turned out to represent a TamB orthologue. This last protein, together with TamA, a protein belonging to the Omp85 family, form a complex that has been proposed to participate in the translocation of autotransporter proteins across the outer membrane (OM). Accordingly, we observed that MapB is required for proper assembly of an autotransporter adhesin in the OM, as most of the autotransporter accumulated in the mutant cell periplasm. Both assessment of the relative amounts of other specific outer membrane proteins (OMPs) and a proteome approach indicated that the absence of MapB did not lead to an extensive alteration in OMP abundance, but to a reduction in the relative amounts of a protein subset, including proteins from the Omp25/31 family. Electron microscopy revealed that ∆mapB cells exhibit multiple anomalies in cell morphology, indicating that the absence of the TamB homologue in B. suis severely affects cell division. Finally, ∆mapB cells were impaired in macrophage infection and showed an attenuated virulence phenotype in the mouse model. Collectively, our results indicate that the role of B. suis TamB homologue is not restricted to participating in the translocation of autotransporters across the OM but that it is essential for OM stability and protein composition and that it is involved in cell envelope biogenesis, a process that is inherently coordinated with cell division.

The BtaF Adhesin Is Necessary for Full Virulence During Respiratory Infection by Brucella suis and Is a Novel Immunogen for Nasal Vaccination Against Brucella Infection.

Brucella enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent Brucella infection. We evaluated the importance of BtaF for respiratory Brucella infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with B. suis M1330 (wild type) and those receiving a ΔbtaF mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. In vitro, these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against B. suis respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of B. suis was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with B. suis by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from B. suis respiratory infection, this adhesin constitutes a promising immunogen against mucosal B. suis infection.

Acidic pH triggers the phosphorylation of the response regulator NtrX in alphaproteobacteria.

Many signaling pathways that control cellular development, cell-cycle progression and nutritional versatility have been studied in Caulobacter crescentus. For example, it was suggested that the response regulator NtrX is conditionally essential for this bacterium and that it might be necessary for responding to a signal produced in phosphate-replete minimal medium. However, such signal has not been identified yet and the role of NtrX in C. crescentus biology remains elusive. Here, using wild-type C. crescentus and a strain with a chromosomally myc-tagged ntrX gene, we demonstrate that high concentrations of phosphate (10 mM) regulate ntrX transcription and the abundance of the protein. We also show that the pH of the medium acts as a switch able to regulate the phosphorylation status of NtrX, promoting its phosphorylation under mildly acidic conditions and its dephosphorylation at neutral pH. Moreover, we demonstrate that the ntrX gene is required for survival in environments with low pH and under acidic stress. Finally, we prove that NtrX phosphorylation is also triggered by low pH in Brucella abortus, a pathogenic alphaproteobacterium. Overall, our work contributes to deepen the general knowledge of this system and provides tools to elucidate the NtrX regulon.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK.

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus.

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.

Cetuximab-mediated cellular cytotoxicity is inhibited by HLA-E membrane expression in colon cancer cells.

Cetuximab, an anti-epidermal growth factor receptor monoclonal antibody, has been shown to increase the median survival of colorectal cancer patients. We previously reported that the expression of HLA-E is significantly increased in primary human colorectal cancer, perhaps contributing to tumour escape from immune surveillance. To establish if HLA-E could be a factor that renders colorectal cancer cells less susceptible to antibody-dependent cellular cytotoxicity (ADCC), in the present study we analysed Cetuximab-mediated cytotoxicity against several colorectal cancer cell lines expressing, or not, HLA-E at the cell surface. We first observed that colorectal cancer cells treated with Cetuximab were killed more efficiently by ADCC. Interestingly, treatment of target cells with recombinant human-beta2-microglobulin inhibits Cetuximab-mediated ADCC through HLA-E membrane stabilization. The specific immunosuppressive role of HLA-E was confirmed using an anti-NKG2A monoclonal antibody, that restored the ability of immune cells to kill their target. This result demonstrates that HLA-E at the cell surface can reliably suppress the ADCC effect. On the other hand, Cetuximab induced a direct growth inhibition but only at high concentrations; furthermore, the CDC effect was quite moderate, and we failed to observe a pro-apoptotic effect. Taking into account that our findings suggest that ADCC activity is the main anti-tumour effect observed at clinically achievable concentrations of Cetuximab at the tumour site, we suggest that determination of HLA-E in colorectal cancer could be relevant to predict success of Cetuximab treatment.