The novel MFS efflux pump SxtP, regulated by the LysR-type transcriptional activator SxtR, is involved in the susceptibility to sulfamethoxazole/trimethoprim (SXT) and the pathogenesis of Acinetobacter baumannii.

Acinetobacter baumannii is a notorious opportunistic pathogen responsible for healthcare-associated infections worldwide. Efflux pumps play crucial roles in mediating antimicrobial resistance, motility, and virulence. In this study, we present the identification and characterization of the new A. baumannii efflux pump SxtP belonging to the MFS superfamily (major facilitator superfamily), along with its associated activator LysR-type transcriptional regulator (LTTR) SxtR, demonstrating their roles in sulfamethoxazole/trimethoprim (also known as co-trimoxazole or SXT) resistance, surface-associated motility and virulence.

Gene expression changes throughout the life cycle allow a bacterial plant pathogen to persist in diverse environmental habitats.

Bacterial pathogens exhibit a remarkable ability to persist and thrive in diverse ecological niches. Understanding the mechanisms enabling their transition between habitats is crucial to control dissemination and potential disease outbreaks. Here, we use Ralstonia solanacearum, the causing agent of the bacterial wilt disease, as a model to investigate pathogen adaptation to water and soil, two environments that act as bacterial reservoirs, and compare this information with gene expression in planta. Gene expression in water resembled that observed during late xylem colonization, with an intriguing induction of the type 3 secretion system (T3SS). Alkaline pH and nutrient scarcity-conditions also encountered during late infection stages-were identified as the triggers for this T3SS induction. In the soil environment, R. solanacearum upregulated stress-responses and genes for the use of alternate carbon sources, such as phenylacetate catabolism and the glyoxylate cycle, and downregulated virulence-associated genes. We proved through gain- and loss-of-function experiments that genes associated with the oxidative stress response, such as the regulator OxyR and the catalase KatG, are key for bacterial survival in soil, as their deletion cause a decrease in culturability associated with a premature induction of the viable but non culturable state (VBNC). This work identifies essential factors necessary for R. solanacearum to complete its life cycle and is the first comprehensive gene expression analysis in all environments occupied by a bacterial plant pathogen, providing valuable insights into its biology and adaptation to unexplored habitats.

Importance of twitching and surface-associated motility in the virulence of Acinetobacter baumannii.

Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.

Direct interaction between RecA and a CheW-like protein is required for surface-associated motility, chemotaxis and the full virulence of Acinetobacter baumannii strain ATCC 17978.

Acinetobacter baumannii is a nosocomial pathogen that causes multi-drug resistant infections mainly in immunocompromised patients. Although this gram-negative species lacks flagella, it is able to move over wet surfaces through a not well characterized type of movement known as surface-associated motility. In this study we demonstrate through the inactivation of the A1S_2813 gene (coding a CheW-like protein) and recA (coding a DNA damage repair and recombination protein) that both genes are involved in the surface-associated motility and chemotaxis of A. baumannii ATCC 17978 strain. In addition, we also point out that the lack of either RecA or CheW-like proteins reduces its virulence in the Caenorhabditis elegans and the Galleria mellonella animal models. Furthermore, we show through co-immunoprecipitation assays that the CheW-like protein and RecA interact and that this interaction is abolished by the introduction of the mutation S97A in one of the domains of CheW-like protein that is structurally conserved in Salmonella enterica and necessary for the RecA-CheW interaction in this bacterial species. Finally, we show that the replacement of the wild-type CheW-like protein by that presenting the S97A mutation impairs surface-associated motility, chemotaxis and virulence of A. baumannii strain ATCC 17978.

Twitching and Swimming Motility Play a Role in Ralstonia solanacearum Pathogenicity.

Ralstonia solanacearum is a bacterial plant pathogen causing important economic losses worldwide. In addition to the polar flagella responsible for swimming motility, this pathogen produces type IV pili (TFP) that govern twitching motility, a flagellum-independent movement on solid surfaces. The implication of chemotaxis in plant colonization, through the control flagellar rotation by the proteins CheW and CheA, has been previously reported in R. solanacearum In this work, we have identified in this bacterium homologues of the Pseudomonas aeruginosapilI and chpA genes, suggested to play roles in TFP-associated motility analogous to those played by the cheW and cheA genes, respectively. We demonstrate that R. solanacearum strains with a deletion of the pilI or the chpA coding region show normal swimming and chemotaxis but altered biofilm formation and reduced twitching motility, transformation efficiency, and root attachment. Furthermore, these mutants displayed wild-type growth in planta and impaired virulence on tomato plants after soil-drench inoculations but not when directly applied to the xylem. Comparison with deletion mutants for pilA and fliC-encoding the major pilin and flagellin subunits, respectively-showed that both twitching and swimming are required for plant colonization and full virulence. This work proves for the first time the functionality of a pilus-mediated pathway encoded by pil-chp genes in R. solanacearum, demonstrating that pilI and chpA genes are bona fide motility regulators controlling twitching motility and its three related phenotypes: virulence, natural transformation, and biofilm formation.IMPORTANCE Twitching and swimming are two bacterial movements governed by pili and flagella. The present work identifies for the first time in the Gram-negative plant pathogen Ralstonia solanacearum a pilus-mediated chemotaxis pathway analogous to that governing flagellum-mediated chemotaxis. We show that regulatory genes in this pathway control all of the phenotypes related to pili, including twitching motility, natural transformation, and biofilm formation, and are also directly implicated in virulence, mainly during the first steps of the plant infection. Our results show that pili have a higher impact than flagella on the interaction of R. solanacearum with tomato plants and reveal new types of cross-talk between the swimming and twitching motility phenotypes: enhanced swimming in bacteria lacking pili and a role for the flagellum in root attachment.

Roles of Efflux Pumps from Different Superfamilies in the Surface-Associated Motility and Virulence of Acinetobacter baumannii ATCC 17978.

Although the relationship between Acinetobacter baumannii efflux pumps and antimicrobial resistance is well documented, less is known about the involvement of these proteins in the pathogenicity of this nosocomial pathogen. In previous work, we identified the AbaQ major facilitator superfamily (MFS) efflux pump and demonstrated its participation in the motility and virulence of A. baumannii In the present study, we examined the role in these processes of A. baumannii transporters belonging to different superfamilies of efflux pumps. Genes encoding known or putative permeases belonging to efflux pump superfamilies other than the MFS were selected, and the corresponding knockouts were constructed. The antimicrobial susceptibilities of these mutants were consistent with previously reported data. In mutants of A. baumannii strain ATCC 17978 carrying inactivated genes encoding the efflux pumps A1S_2736 (resistance nodulation division [RND]), A1S_3371 (multidrug and toxic compound extrusion [MATE]), and A1S_0710 (small multidrug resistance [SMR]), as well as the newly described ATP-binding cassette (ABC) permeases A1S_1242 and A1S_2622, both surface-associated motility and virulence were reduced compared to the parental strain. However, inactivation of the genes encoding the known ABC permeases A1S_0536 and A1S_1535, the newly identified putative ABC permeases A1S_0027 and A1S_1057, or the proteobacterial antimicrobial compound efflux (PACE) transporters A1S_1503 and A1S_2063 had no effects on bacterial motility or virulence. Our results demonstrate the involvement of antimicrobial transporters belonging at least to five of the six known efflux pump superfamilies in both surface-associated motility and virulence in A. baumannii ATCC 17978.

Heterozygous STUB1 mutation causes familial ataxia with cognitive affective syndrome (SCA48).

OBJECTIVE: To describe a new spinocerebellar ataxia (SCA48) characterized by early cerebellar cognitive-affective syndrome (CCAS) and late-onset SCA. METHODS: This is a descriptive study of a family that has been followed for more than a decade with periodic neurologic and neuropsychological examinations, MRI, brain SPECT perfusion, and genetic analysis. Whole exome sequencing was performed in 3 affected and 1 unaffected family member and subsequently validated by linkage analysis of chromosome 16p13.3. RESULTS: Six patients fully developed cognitive-affective and complete motor cerebellar syndrome associated with vermian and hemispheric cerebellar atrophy, suggesting a continuum from a dysexecutive syndrome slowly evolving to a complete and severe CCAS with late truncal ataxia. Three presymptomatic patients showed focal cerebellar atrophy in the vermian, paravermian, and the medial part of cerebellar lobes VI and VII, suggesting that cerebellar atrophy preceded the ataxia, and that the neurodegeneration begins in cerebellar areas related to cognition and emotion, spreading later to the whole cerebellum. Among the candidate variants, only the frameshift heterozygous c.823_824delCT STUB1 (p.L275Dfs*16) pathogenic variant cosegregated with the disease. The p.L275Dfs*16 heterozygous STUB1 pathogenic variant leads to neurodegeneration and atrophy in cognition- and emotion-related cerebellar areas and reinforces the importance of STUB1 in maintaining cognitive cerebellar function. CONCLUSIONS: We report a heterozygous STUB1 pathogenic genetic variant causing dominant cerebellar ataxia. Since recessive mutations in STUB1 gene have been previously associated with SCAR16, these findings suggest a previously undescribed SCA locus (SCA48; MIM# 618093).

Functional Characterization of AbaQ, a Novel Efflux Pump Mediating Quinolone Resistance in Acinetobacter baumannii.

Acinetobacter baumannii has emerged as an important multidrug-resistant nosocomial pathogen. In previous work, we identified a putative MFS transporter, AU097_RS17040, involved in the pathogenicity of A. baumannii (M. Pérez-Varela, J. Corral, J. A. Vallejo, S. Rumbo-Feal, G. Bou, J. Aranda, and J. Barbé, Infect Immun 85:e00327-17, 2017, https://doi.org/10.1128/IAI.00327-17). In this study, we analyzed the susceptibility to diverse antimicrobial agents of A. baumannii cells defective in this transporter, referred to as AbaQ. Our results showed that AbaQ is mainly involved in the extrusion of quinolone-type drugs in A. baumannii.

Mutations in the ß-Subunit of the RNA Polymerase Impair the Surface-Associated Motility and Virulence of Acinetobacter baumannii.

Acinetobacter baumannii is a major cause of antibiotic-resistant nosocomial infections worldwide. In this study, several rifampin-resistant spontaneous mutants obtained from the A. baumannii ATCC 17978 strain that differed in their point mutations in the rpoB gene, encoding the ß-subunit of the RNA polymerase, were isolated. All the mutants harboring amino acid substitutions in position 522 or 540 of the RpoB protein were impaired in surface-associated motility and had attenuated virulence in the fertility model of Caenorhabditis elegans The transcriptional profile of these mutants included six downregulated genes encoding proteins homologous to transporters and metabolic enzymes widespread among A. baumannii clinical isolates. The construction of knockout mutants in each of the six downregulated genes revealed a significant reduction in the surface-associated motility and virulence of four of them in the A. baumannii ATCC 17978 strain, as well as in the virulent clinical isolate MAR002. Taken together, our results provide strong evidence of the connection between motility and virulence in this multiresistant nosocomial pathogen.

Novobiocin Inhibits the Antimicrobial Resistance Acquired through DNA Damage-Induced Mutagenesis in Acinetobacter baumannii.

Acinetobacter baumannii, a worldwide emerging nosocomial pathogen, acquires antimicrobial resistances in response to DNA-damaging agents, which increase the expression of multiple error-prone DNA polymerase components. Here we show that the aminocoumarin novobiocin, which inhibits the DNA damage response in Gram-positive bacteria, also inhibits the expression of error-prone DNA polymerases in this Gram-negative multidrug-resistant pathogen and, consequently, its potential acquisition of antimicrobial resistance through DNA damage-induced mutagenesis.

New subtype of spinocerebellar ataxia with altered vertical eye movements mapping to chromosome 1p32.

IMPORTANCE: To provide clinical and genetic diagnoses for patients' conditions, it is important to identify and characterize the different subtypes of spinocerebellar ataxia (SCA). OBJECTIVE: To clinically and genetically characterize a Spanish kindred with pure SCA presenting with altered vertical eye movements. DESIGN Family study of ambulatory patients. Electro-oculographic and genetics studies were performed in 2 referral university centers. SETTING: Primary care institutional center in Spain. PARTICIPANTS: Thirty-six participants from a large Spanish kindred were clinically examined, and 33 family members were genetically examined. Detailed clinical data were obtained from 9 affected relatives. Two ataxic siblings and 2 asymptomatic family members were examined using an enhanced clinical protocol for a follow-up period of 7 years. MAIN OUTCOMES AND MEASURES: High-density genome-wide single-nucleotide polymorphism arrays, along with microsatellite analysis, and genetic linkage studies were performed. Whole-exome sequencing was used for 2 affected relatives. For most patients, the initial symptoms included falls, dysarthria, or clumsiness followed by a complete cerebellar syndrome. For all 9 affected relatives, we observed altered vertical eye movements, as initial ocular signs for 3 of them and for the 2 asymptomatic family members, all having inherited the risk haplotype. Neuroimaging showed isolated cerebellar atrophy. RESULTS: Initial genome-wide linkage analysis revealed suggestive linkage to chromosome 1p32. Multipoint analysis and haplotype reconstruction further traced this SCA locus to a 0.66-cM interval flanked by D1S200 and D1S2742 (z(max) = 6.539; P < .0001). The causative mutation was unidentified by exome sequencing. CONCLUSIONS AND RELEVANCE: We report a new subtype of SCA presenting in patients as slow progressing ataxia with altered vertical eye movements linked to a 11-megabase interval on 1p32. The Human Genome Nomenclature Committee has assigned this subtype of ataxia the designation SCA37.

Genotype of an individual single nucleotide polymorphism regulates DNA methylation at the TRPC3 alternative promoter.

A fundamental challenge in the post-genomics era is to understand how genetic variants can influence phenotypic variability and disease. Recent observations from a number of studies have highlighted a mechanism by which common genetic polymorphisms can influence DNA methylation, a major epigenetic silencing mechanism. We report that the alternative promoter of the human TRPC3 gene is regulated by allelic DNA methylation, dictated by the genotype of a single base pair polymorphism, rs13121031 located within the promoter CpG island. The common G allele is associated with high levels of methylation, while the less prevalent C allele is unmethylated. This methylation profile is observed in many tissue types, despite the expression of TRPC3 being restricted to brain and heart. TRPC3 is prominently expressed in the hindbrain, and a heterozygous brain sample showed modest skewing according to the allelic methylation, with preferential expression from the C allele. The TRPC3 gene encodes a transient receptor potential channel that has been implicated in cerebellar ataxia and heart hypertrophy. The genotype-frequencies of rs13121031 were determined in cohorts of ataxia patients and in individuals with cardiac hypertrophy. These analyses revealed a statistical trend for the rare unmethylated homozygous C genotype to be present at a higher frequency in idiopathic ataxia patients (Fisher's test p=0.06), but not in those patients with known mutations (Fisher's test p=0.55) or in individuals with heart disease (Fisher's test p=0.807), when compared to a control population. Our results suggest that the TRPC3 alternative promoter is a methylation quantitative-trait locus that may be involved in modulating the ataxia phenotype.

Cerebellar ataxia with coenzyme Q10 deficiency: diagnosis and follow-up after coenzyme Q10 supplementation.

UNLABELLED: Our aim was to report a new case with cerebellar ataxia associated with coenzyme Q10 (CoQ) deficiency, the biochemical findings caused by this deficiency and the response to CoQ supplementation. PATIENT: A 12-year-old girl presenting ataxia and cerebellar atrophy. BIOCHEMICAL STUDIES: Coenzyme Q10 in muscle was analysed by HPLC with electrochemical detection and mitochondrial respiratory chain (MRC) enzyme activities by spectrophotometric methods. CoQ biosynthesis in fibroblasts was assayed by studying the incorporation of radiolabeled 4-hydroxy[U 14C] benzoic acid by HPLC with radiometric detection. RESULTS: Mitochondrial respiratory chain enzyme analysis showed a decrease in complex I + III and complex II + III activities. CoQ concentration in muscle was decreased (56 nmol/g of protein: reference values: 157-488 nmol/g protein). A reduced incorporation of radiolabeled 4-hydroxy[U- 14C] benzoic acid was observed in the patient (19% of incorporation respect to the median control values). After 16 months of CoQ supplementation, the patient is now able to walk unaided and cerebellar signs have disappeared. CONCLUSIONS: Cerebellar ataxia associated with CoQ deficiency in our case might be allocated in the transprenylation pathway or in the metabolic steps after condensation of 4-hydroxybenzoate and the prenyl side chain of CoQ. Clinical improvement after CoQ supplementation was remarkable, supporting the importance of an early diagnosis of this kind of disorders.

Giant SCA8 alleles in nine children whose mother has two moderately large ones.

We report here a family in which each of nine children has inherited giant SCA8 CTG expansions from a homozygous mother who has two moderately large SCA8 CTG alleles. In contrast, three homozygous male individuals and a case of coexistence of two expansions of the FRDA gene and one of SCA8, all of them with moderately large alleles, have transmitted their respective SCA8 expanded alleles with minor changes, as usually occurs in heterozygous male transmissions.

Spinocerebellar ataxia type 2 with Levodopa-responsive parkinsonism culminating in motor neuron disease.

We describe an exceptional spinocerebellar ataxia type 2 (SCA2) phenotype combining cerebellar ataxia, levodopa-responsive parkinsonism, and motor neuron symptoms. We conclude that motor neuron symptoms and signs may be a striking manifestation in SCA2, masking pre-existing cerebellar and extrapyramidal semeiology.