Coalition for Health and Gender Equity (CHANGE)—a protocol for a global cross-sectional survey of health and gender equity in rheumatology

Lay Summary: What does this mean for patients? The CHANGE Study, led by a team of rheumatology professionals worldwide, is working to make health care more equal for everyone. We are focusing on challenges faced by rheumatologists, such as fair pay and career opportunities. To understand these issues better, the team is gathering information through a global survey of rheumatology professionals. The goal is to find out why there are differences and come up with solutions. Ultimately, the aim is to create a fair and inclusive environment in rheumatology, ensuring that everyone has the same chances to grow in their careers, regardless of their gender. The findings of the study will help to create better guidelines, promoting fairness and equality for health-care professionals in rheumatology

The Pathogenic Properties of a Novel and Conserved Gene Product, KerV, in Proteobacteria

Identification of novel virulence factors is essential for understanding bacterial pathogenesis and designing antibacterial strategies. In this study, we uncover such a factor, termed KerV, in Proteobacteria. Experiments carried out in a variety of eukaryotic host infection models revealed that the virulence of a Pseudomonas aeruginosa kerV null mutant was compromised when it interacted with amoebae, plants, flies, and mice. Bioinformatics analyses indicated that KerV is a hypothetical methyltransferase and is well-conserved across numerous Proteobacteria, including both well-known and emerging pathogens (e.g., virulent Burkholderia, Escherichia, Shigella, Vibrio, Salmonella, Yersinia and Brucella species). Furthermore, among the 197 kerV orthologs analyzed in this study, about 89% reside in a defined genomic neighborhood, which also possesses essential DNA replication and repair genes and detoxification gene. Finally, infection of Drosophila melanogaster with null mutants demonstrated that KerV orthologs are also crucial in Vibrio cholerae and Yersinia pseudotuberculosis pathogenesis. Our findings suggested that KerV has a novel and broad significance as a virulence factor in pathogenic Proteobacteria and it might serve as a new target for antibiotic drug design

Longer-term effectiveness of a heterologous coronavirus disease 2019 (COVID-19) vaccine booster in healthcare workers in Brazil

Abstract Objective: To compare the long-term vaccine effectiveness between those receiving viral vector [Oxford-AstraZeneca (ChAdOx1)] or inactivated viral (CoronaVac) primary series (2 doses) and those who received an mRNA booster (Pfizer/BioNTech) (the third dose) among healthcare workers (HCWs). Methods: We conducted a retrospective cohort study among HCWs (aged ≥18 years) in Brazil from January 2021 to July 2022. To assess the variation in the effectiveness of booster dose over time, we estimated the effectiveness rate by taking the log risk ratio as a function of time. Results: Of 14,532 HCWs, coronavirus disease 2019 (COVID-19) was confirmed in 56.3% of HCWs receiving 2 doses of CoronaVac vaccine versus 23.2% of HCWs receiving 2 doses of CoronaVac vaccine with mRNA booster (P < .001), and 37.1% of HCWs receiving 2 doses of ChAdOx1 vaccine versus 22.7% among HCWs receiving 2 doses of ChAdOx1 vaccine with mRNA booster (P < .001). The highest vaccine effectiveness with mRNA booster was observed 30 days after vaccination: 91% for the CoronaVac vaccine group and 97% for the ChAdOx1 vaccine group. Vacine effectiveness declined to 55% and 67%, respectively, at 180 days. Of 430 samples screened for mutations, 49.5% were SARS-CoV-2 delta variants and 34.2% were SARS-CoV-2 omicron variants. Conclusions: Heterologous COVID-19 vaccines were effective for up to 180 days in preventing COVID-19 in the SARS-CoV-2 delta and omicron variant eras, which suggests the need for a second booster

Genes involved with Pseudomonas aeruginosa PA14 pathogenicity: characterization of the promoter regions and virulence assays in the Dictyostelium discoideum host model

Pseudomonas aeruginosa é uma gamaproteobactéria ubíqua capaz de infectar indivíduos imunocomprometidos e causar infecções hospitalares. Entre duas repetições diretas situadas na ilha de patogenicidade PAPI-1 da linhagem PA14, encontram-se dois grupos de genes transcritos em direções opostas. O primeiro, composto de pvrS, pvrR, rcsC e rcsB, codifica proteínas de sistemas de dois componentes e está relacionado com virulência, enquanto o segundo compreende cinco genes (cupD1-D5) e codifica uma fímbria do tipo chaperoneusher, com alta similaridade com o grupo cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Fímbrias da mesma família são importantes na patogenicidade de outras bactérias. Com o objetivo de estudar a relação entre esses dois grupos de genes, procurou-se caracterizar sua organização por ensaios de RT-PCR, que possibilitaram observar a disposição dos genes dos sistemas de dois componentes em dois operons distintos (pvrRS e rcsCB) e, pelo menos, cupD1-cupD2 como uma unidade transcricional, com indícios apontando para a organização de cupD1-D5 em um único operon. Os inícios de transcrição e as regiões promotoras de cada operon foram caracterizados por experimentos de RACE 5 e extensão de oligonucleotídeo marcado em busca de seqüências relevantes para a ativação da expressão desses genes. Visando investigar a regulação da expressão da fímbria CupD pelos sistemas de dois componentes codificados pelos genes adjacentes, foram realizados ensaios de qRT-PCR, os quais mostraram uma menor expressão de cupD na linhagem mutante para rcsB. RcsB apresenta um domínio de ligação a DNA e, apesar da falta de sucesso em ensaios de ligação dessa proteína à região promotora de cupD, os dados obtidos por qRT-PCR 1 indicam fortemente que essa proteína funciona como um ativador de transcrição dos genes da fímbria. Corroborando esses achados, somente quando se utilizou RNA extraído de P. aeruginosa PA14 superexpressando RcsB foi possível visualizar no gel a banda referente ao início de transcrição de cupD1-D2 no ensaio de extensão de oligonucleotídeo. Ao contrário do efeito positivo de RcsB observado na transcrição de cupD1, cupD2 e cupD5, a histidina quinase RcsC atua negativamente na expressão dos genes da fímbria, sugerindo que, nesse caso, sua atividade predominante sobre RcsB seja a de fosfatase. PvrS e PvrR parecem atuar de forma indireta e positiva sobre cupD. Como um segundo objetivo do trabalho, ensaios de virulência de P. aeruginosa no hospedeiro-modelo Dictyostelium discoideum foram otimizados, com o estabelecimento de uma técnica para se testar alguns genes estudados no laboratório que podem ser relevantes para a virulência dessa bactéria. Resultados desses ensaios confirmaram a atenuação de mutantes para uma suposta metiltransferase, já observada em modelos de planta, camundongo e drosófila. Em conjunto, os resultados desse trabalho tornam-se informações valiosas para serem usadas na pesquisa de controle de infecções por P. aeruginosa, que depende de fímbrias para colonizar com sucesso superfícies abióticas que servem de veículo de disseminação desse agente infeccioso e para que as bactérias possam persistir no organismo do hospedeiro.Pseudomonas aeruginosa is a ubiquitous gammaproteobacteria able to infect immunocompromised individuals and to cause nosocomial infections. Between two direct repeats in the PAPI-1 pathogenicity island present in strain PA14, there are two gene clusters transcribed in opposite directions. The first, composed of pvrS, pvrR, rcsC and rcsB, encodes two-component systems proteins and is implicated in virulence, whereas the second comprises five genes (cupD1-D5) encoding a chaperone-usher fimbria with high similarity to cupA, a gene cluster involved in biofilm formation in other strains of P. aeruginosa. Fimbriae belonging to the same family of Cup are related to pathogenicity of other bacteria. In order to study the relationship between these two clusters, the organization of the genes in operons was characterized using RT-PCR, which results lead to the conclusion that the twocomponent systems genes are arranged into two different operons (pvrSR and rcsCB) and that cupD1-D2 share the same promoter, with some evidences that the operons extends from cupD1 to cupD5. The transcription start sites and the promoter regions of each operon were characterized with RACE 5 and primer extension assays to look for sequences that could be relevant to the activation of expression of these genes. Quantitative RT-PCR assays were carried out to investigate whether the expression of CupD fimbriae is regulated by the twocomponent systems encoded by the adjacent genes and the results showed a lower expression of cupD in the rcsB mutant strain than in the wild-type. RcsB bears a DNA-binding domain and, although our assays of DNA-protein interactions have failed, data obtained by qRT-PCR 1 strongly indicate that this protein functions as a transcription activator of fimbrial genes. These findings were corroborated by the primer extension assay, in which the band corresponding to the transcriptional start of cupD1-D2 was visible only when the reaction was performed with the RNA extracted of P. aeruginosa overexpressing RcsB. Unlike the effect observed for RcsB in cupD1, cupD2 and cupD5 transcription, the histidine quinase RcsC acts negatively in the fimbrial genes expression, suggesting that it might function predominantly as a RcsB phosphatase. PvrS and PvrR seem to regulate cupD positively and indirectly. As a second aim of this work, virulence assays of P. aeruginosa in the model host Dictyostelium discoideum were optimized, and a technique for testing genes studied in the laboratory that could be important for Pseudomonas virulence was established. These assays confirmed the attenuation-in-virulence of strains mutant in a putative methyltransferase gene, as observed before in plant, mouse and drosophila models. The results obtained in this work may contribute to P. aeruginosa infection control research, since this bacterium depends on fimbriae to successfully colonize abiotic surfaces that act as a dissemination vehicle, and to allow the bacteria to persist into the host organism

Characterization ofthe ECF sigma fator &#963;x overexpression in Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama muito versátil, capaz de colonizar ambientes variados e infectar hospedeiros filogeneticamente distintos, incluindo humanos imunocomprometidos. Os fatores sigma de função extracitoplasmática (ECF) são membros de sistemas de sinalização de superfície celular (CSS), abundantes em P. aeruginosa. Vinte genes codificando fatores sigma ECF estão presentes nos genomas sequenciados de P. aeruginosa, a maioria fazendo parte de sistemas TonB relacionados à captação de ferro. Neste trabalho, seis fatores sigma pobremente caracterizados foram superexpressos na linhagem PA14 a partir de um promotor induzível por arabinose para investigar seu papel na expressão dos sistemas de dois componentes PvrSR e RcsCB, que atuam na regulação da fímbria CupD, além de sua influência no crescimento de culturas de P. aeruginosa. Não foi observado efeito positivo de nenhum dos fatores sigma testados na expressão dos sistemas de dois componentes e a superexpressão de cinco deles tampouco levou a qualquer alteração no crescimento, porém a produção de piocianina foi alterada na superexpressão de PA14_55550 e a superexpressão de PA14_26600 e PA14_46810 levou a um discreto aumento no início da formação de biofilme em PA14. Por outro lado, culturas superexpressando &#963;x (ALB04) apresentaram um perfil alterado de lipopolissacarídeo e uma curva de crescimento bifásica, alcançando precocemente uma fase estacionária seguida de uma recuperação do crescimento até uma segunda fase estacionária. Durante a primeira fase estacionária, a maior parte das células aumenta de tamanho e morre, mas as células remanescentes retornam à morfologia selvagem e seguem para a segunda fase de crescimento exponencial. Isso não acontece devido a mutações compensatórias, uma vez que células coletadas de pontos tardios da curva e diluídas em meio novo repetem este comportamento. Apesar de trabalhos com a linhagem PAO1 associarem &#963;x à transcrição de oprF, que codifica a principal porina não específica de Pseudomonas, nas condições dos nossos ensaios em PA14 a expressão dessa porina não foi induzida pela superexpressão de &#963;x. Assim, os efeitos observados nessa superexpressão também não podem ser atribuídos a OprF. A transcrição de oprF em PA14 mostrou-se majoritariamente dependente da região promotora a que se atribui a ligação de &#963;70, ao contrário dos relatos na literatura da dependência da região de ligação a &#963;x. Análises proteômicas foram realizadas para investigar os elementos envolvidos nesses efeitos de superexpressão de &#963;x, o que revelou a indução de diversas enzimas envolvidas na via de biossíntese de ácidos graxos. As células superexpressando &#963;x apresentam uma maior proporção de ácidos hexadecanoico (C16) e hexadecenoico (C16:1) e dados de anisotropia mostram uma maior fluidez da(s) membrana(s). Este trabalho é o primeiro relato de um fator sigma ECF envolvido em biossíntese de lipídeos em P. aeruginosa.Pseudomonas aeruginosa is a very versatile gammaproteobacteria, able to colonize different environments and to infect phylogenetically distinct hosts, including immunocompromised humans. The extracytoplasmic function sigma factors (ECFs) are members of cell signaling systems (CSS), abundant in P. aeruginosa. Twenty genes coding for ECF sigma factors are present in the sequenced genomes of P. aeruginosa, most of them being part of TonB systems related to iron uptake. In this work, six poorly characterized sigma factors were overexpressed in strain PA14 from an arabinose inducible promoter to investigate their role in the expression of the two-component systems PvrSR and RcsCB, which regulates CupD fimbria, and their influence in P. aeruginosa cultures growth. None of the tested sigma factors led to two-component systems upregulation and overexpression of five of them caused no change in the growth profile, but pyocyanin production was altered in PA14_55550 overexpression and PA14_26600 and PA14_46810 overexpression led to a slight increase in biofilm initiation in PA14. By the other side, cultures overexpressing &#963;x (ALB04) presented an altered lipopolysaccharide profile and a biphasic growth curve, reaching an early stationary phase followed by a growth resuming untill a second stationary phase. During the early stationary phase, most cells swells and dies, but the remaining cells return to wild type morphology and proceed to the second exponential phase of growth. This is not due to compensatory mutations, since cells collected from late points of the curve and diluted in fresh medium repeat this behavior. Although studies with strain PAO1 associate &#963;x with transcription of oprF, encoding the major nonspecific porin of Pseudomonas, under our experiments conditions with PA14, this porin expression is not induced by &#963;x overexpression. Thus, the effects observed in this overexpression cannot be attributed to OprF. Transcription of oprF in PA14 proved to be mainly controlled by the &#963;70-dependent promoter region instead of the &#963;x-dependent promoter region reported in the literature. Proteomic analyses were performed to investigate the elements involved in these effects of &#963;x overexpression, which revealed the induction of several enzymes involved in fatty acids biosynthesis. Cells overexpressing &#963;x exhibit a greater proportion of hexadecanoic (C16) and hexadecenoic (C16: 1) acids and anisotropy data show higher fluidity of the membrane (s). This work is the first report of an ECF sigma factor involved in lipid biosynthesis in P. aeruginosa

A novel role for an ECF sigma factor in fatty acid biosynthesis and membrane fluidity in Pseudomonas aeruginosa.

International audienceExtracytoplasmic function (ECF) sigma factors are members of cell-surface signaling systems, abundant in the opportunistic pathogen Pseudomonas aeruginosa. Twenty genes coding for ECF sigma factors are present in P. aeruginosa sequenced genomes, most of them being part of TonB systems related to iron uptake. In this work, poorly characterized sigma factors were overexpressed in strain PA14, in an attempt to understand their role in the bacterium's physiology. Cultures overexpressing SigX displayed a biphasic growth curve, reaching stationary phase earlier than the control strain, followed by subsequent growth resumption. During the first stationary phase, most cells swell and die, but the remaining cells return to the wild type morphology and proceed to a second exponential growth. This is not due to compensatory mutations, since cells recovered from late time points and diluted into fresh medium repeated this behavior. Swollen cells have a more fluid membrane and contain higher amounts of shorter chain fatty acids. A proteomic analysis was performed to identify differentially expressed proteins due to overexpression of sigX, revealing the induction of several fatty acid synthesis (FAS) enzymes. Using qRT-PCR, we showed that at least one isoform from each of the FAS pathway enzymes were upregulated at the mRNA level in the SigX overexpressing strain thus pointing to a role for this ECF sigma factor in the FAS regulation in P. aeruginosa

Pseudomonas aeruginosa PA14 cupD transcription is activated by the RcsB response regulator, but repressed by its putative cognate sensor RcsC

The opportunistic pathogen Pseudomonas aeruginosa PA14 possesses four fimbrial cup clusters, which may confer the ability to adapt to different environments. cupD lies in the pathogenicity island PAPI-1 next to genes coding for a putative phosphorelay system composed of the hybrid histidine kinase RcsC and the response regulator RcsB. The main focus of this work was the regulation of cupD at the mRNA level. It was found that the HN-S-like protein MvaT does not exert a strong influence on cupD transcript levels, as it does for cupA. cupD transcription is higher in cultures grown at 28 degrees C, which agrees with a cupD mutant presenting attenuated virulence only in a plant model, but not in a mouse model of infection. Whereas an rcsC in-frame deletion mutant presented higher levels of cupD mRNA, rcsB deletion had the opposite effect. Accordingly, overexpression of RcsB increased the levels of cupD transcription, and promoted biofilm formation and the appearance of fimbriae. A single transcription start site was determined for cupD and transcription from this site was induced by RcsB. A motif similar to the enterobacterial RcsB/RcsA-binding site was detected adjacent to the -35 region, suggesting that this could be the RcsB-binding site. Comparison of P. aeruginosa and Escherichia coli Rcs may provide insights into how similar systems can be used by different bacteria to control gene expression and to adapt to various environmental conditions.FAPESP Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)CNPq Conselho Nacional de Desenvolvimento Cientifico e TecnologicoConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Lower levels of overexpression and natural induction of sig<i>X</i> induce target genes.

<p><b>A</b>. Arabinose was added to ALB04 cultures at exponential phase and samples (+Ara) were collected after 15, 30 and 180 minutes for RNA extraction and qRT-PCR of sig<i>X</i>, accA and <i>fabH3</i>. Control samples were taken at the same time points (-Ara). All values are shown as relative to t=0 for each treatment, set as 1. <b>B</b>. β-galactosidase activity assay of sig<i>X</i> and <i>fabH3</i>-<i>lacZ</i> promoter fusions in the wild-type background (PA14::sig<i>X</i>_<i>lacZ</i> and PA14::<i>fabH3_lacZ</i>) in LB with 171 mM NaCl or without NaCl. The values plotted are averages of biological triplicates and error bars represent standard deviations.</p

SigX overexpression alters growth and morphology of <i>P. aeruginosa</i>.

<p><b>A</b>. Growth curve of sig<i>X</i> overexpressing strain (ALB04) and control (ALB01) in LB with 50 mg/mL gentamycin and 0.2% of arabinose at 37°C and 240 rpm. <b>B</b>. Survival was measured by counting colony forming units (CFU) along the growth curve. <b>C</b>. Phase contrast and fluorescence microscopy. In both panels, the column on the left shows the cells as seen in phase contrast microscopy, while the column on the right depicts fluorescence microscopy of the cultures previously stained with LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen), where membrane damaged cells (dead) are colored in red. All pictures are at the same scale. D. Flow cytometry analysis of LIVE/DEAD stained cells. The values plotted in A and B are means of three biological replicates and error bars represent standard deviation. Flow cytometry was carried out with biological duplicates in two different occasions and values shown are representative of one experiment.</p