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172
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"Veiga, Helena"
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A new regulator of the Staphylococcus aureus peptidoglycan hydrolase Sle1
Regulation of peptidoglycan hydrolases is crucial for bacterial cell integrity, growth and division. In the bacterial pathogen Staphylococcus aureus , the amidase Sle1 is a key autolysin required for septum splitting and daughter cell separation. Through genetic suppressor screening, we have identified CxaR, a previously uncharacterized protein, as a novel negative regulator of Sle1. In the absence of CxaR, cellular levels of Sle1 increase nearly ten-fold, resulting in premature splitting of the division septum and increased cell lysis during exponential growth. CxaR localizes to the division septum, late in septum synthesis, and this localization requires both the divisome protein FtsK and the ClpX component of the ClpXP proteolytic machinery. We propose that CxaR promotes ClpXP-mediated degradation of Sle1 towards the end of the cell cycle.
Journal Article
Synthetic antimicrobial peptides as enhancers of the bacteriolytic action of staphylococcal phage endolysins
Bacteriophage endolysins degrade the bacterial cell wall and are therefore considered promising antimicrobial alternatives to fight pathogens resistant to conventional antibiotics. Gram-positive bacteria are usually considered easy targets to exogenously added endolysins, since their cell walls are not shielded by an outer membrane. However, in nutrient rich environments these bacteria can also tolerate endolysin attack if they keep an energized cytoplasmic membrane. Hence, we have hypothesized that the membrane depolarizing action of antimicrobial peptides (AMPs), another attractive class of alternative antibacterials, could be explored to overcome bacterial tolerance to endolysins and consequently improve their antibacterial potential. Accordingly, we show that under conditions supporting bacterial growth,
Staphylococcus aureus
becomes much more susceptible to the bacteriolytic action of endolysins if an AMP is also present. The bactericidal gain resulting from the AMP/endolysin combined action ranged from 1 to 3 logs for different
S. aureus
strains, which included drug-resistant clinical isolates. In presence of an AMP, as with a reduced content of cell wall teichoic acids, higher endolysin binding to cells is observed. However, our results indicate that this higher endolysin binding alone does not fully explain the higher susceptibility of
S. aureus
to lysis in these conditions. Other factors possibly contributing to the increased endolysin susceptibility in presence of an AMP are discussed.
Journal Article
Cell shape dynamics during the staphylococcal cell cycle
Staphylococcus aureus
is an aggressive pathogen and a model organism to study cell division in sequential orthogonal planes in spherical bacteria. However, the small size of staphylococcal cells has impaired analysis of changes in morphology during the cell cycle. Here we use super-resolution microscopy and determine that
S. aureus
cells are not spherical throughout the cell cycle, but elongate during specific time windows, through peptidoglycan synthesis and remodelling. Both peptidoglycan hydrolysis and turgor pressure are required during division for reshaping the flat division septum into a curved surface. In this process, the septum generates less than one hemisphere of each daughter cell, a trait we show is common to other cocci. Therefore, cell surface scars of previous divisions do not divide the cells in quadrants, generating asymmetry in the daughter cells. Our results introduce a need to reassess the models for division plane selection in cocci.
Staphylococci are spherical bacteria that divide in sequential orthogonal planes. Here, the authors use super-resolution microscopy to show that staphylococcal cells elongate before dividing, and that the division septum generates less than one hemisphere of each daughter cell, generating asymmetry.
Journal Article
A CRISPRi-based genetic resource to study essential Staphylococcus aureus genes
We have optimized a clustered regularly interspaced short palindromic repeat (CRISPR) interference system to facilitate gene knockdown in the Gram-positive bacterial pathogen Staphylococcus aureus . Our approach used a CRISPRi system derived from Streptococcus pyogenes, which involves the co-expression of the dcas9 gene encoding a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). In our system, dcas9 is expressed from a single copy in the chromosome of methicillin-resistant S. aureus strains COL or JE2, under the control of a tightly regulated promoter, inducible by anhydrotetracycline. The sgRNAs are expressed from a replicative plasmid under the control of a constitutively active promoter. This system enables efficient, inducible, knockdown of both essential and non-essential genes. Using this approach, we constructed the Lisbon CRISPRi Mutant Library comprising 261 strains, in the JE2 background, containing sgRNAs targeting 200 essential genes/operons. This library facilitates the study of the function of essential S. aureus genes and is complementary to the Nebraska Transposon Mutant Library, which consists of nearly 2,000 strains, each carrying a transposon insertion within a non-essential gene. The availability of these two libraries will facilitate the study of S. aureus pathogenesis and biology. Staphylococcus aureus is an important clinical pathogen that causes a high number of antibiotic-resistant infections. The study of S. aureus biology, and particularly of the function of essential proteins, is of particular importance to develop new approaches to combat this pathogen. We have optimized a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system that allows efficient targeting of essential S. aureus genes. Furthermore, we have used that system to construct a library comprising 261 strains, which allows the depletion of essential proteins encoded by 200 genes/operons. This library, which we have named Lisbon CRISPRi Mutant Library, should facilitate the study of S. aureus pathogenesis and biology.
Journal Article
Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus
The cell wall of Staphylococcus aureus is characterized by an extremely high degree of cross-linking within its peptidoglycan (PGN). Penicillin-binding protein 4 (PBP4) is required for the synthesis of this highly cross-linked peptidoglycan. We found that wall teichoic acids, glycopolymers attached to the peptidoglycan and important for virulence in Gram-positive bacteria, act as temporal and spatial regulators of PGN metabolism, controlling the level of cross-linking by regulating PBP4 localization. PBP4 normally localizes at the division septum, but in the absence of wall teichoic acids synthesis, it becomes dispersed throughout the entire cell membrane and is unable to function normally. As a consequence, the peptidoglycan of TagO null mutants, impaired in wall teichoic acid biosynthesis, has a decreased degree of cross-linking, which renders it more susceptible to the action of lysozyme, an enzyme produced by different host organisms as an initial defense against bacterial infection.
Journal Article
Genetic insights from a Brazilian cohort of aortopathies through targeted next-generation sequencing and FBN1 direct sequencing
Thoracic aortic diseases (or aortopathies) result from complex interactions between genetic and hemodynamic factors. Often clinically silent, these diseases can lead to lethal complications such as aortic dissection or rupture. This study focused on a Brazilian cohort of 79 individuals with thoracic aortic diseases and explored genetic factors through targeted next-generation sequencing (tNGS) of 15 priority genes and
FBN1
direct sequencing. The majority of individuals had nonsyndromic aortopathy, with eight diagnosed with Marfan syndrome (MFS). Pathogenic or likely pathogenic variants (PV/LPV) were found in five genes, namely,
FBN1
,
ACTA2
,
TGFBR2
,
MYLK
, and
SMAD3
. Notably, novel variants in
FBN1
were identified that contributed to Marfan-like phenotypes. The diagnostic yield for isolated aortopathies was 7.1%, which increased to 55.5% for syndromic cases. Variants of uncertain significance (VUS) were identified, emphasizing the need for further research and familial investigations to refine variant classifications. This study provides valuable insights into the genetic landscape of aortopathies in Brazil, aiding early diagnosis and personalized management.
Journal Article
The role of GpsB in Staphylococcus aureus cell morphogenesis
Staphylococcus aureus is a Gram-positive clinical pathogen, which is currently the second cause of death by antibiotic-resistant infections worldwide. For decades, S. aureus cells were thought to be spherical and lack the ability to undergo elongation. However, super-resolution microscopy techniques allowed us to observe the minor morphological changes that occur during the cell cycle of this pathogen, including cell elongation. S. aureus elongation is not required for normal growth in laboratory conditions. However, it seems to be essential in the context of some infections, such as osteomyelitis, during which S. aureus cells apparently elongate to invade small channels in the bones. In this work, we uncovered new determinants required for S. aureus cell elongation. In particular, we show that GpsB has an important role in the spatio-temporal regulation of PBP2 and PBP4, two proteins involved in peptidoglycan synthesis, contributing to the maintenance of the correct cell morphology in S. aureus .
Journal Article
Sociodemographic Associations and COVID-19 Symptoms Following One Year of Molecular Screening for SARS-CoV-2 Among Healthcare Workers
Background: During the COVID-19 pandemic, high rates of infection with SARS-CoV-2 were reported in healthcare workers (HCWs), among whom asymptomatic individuals had high potential to spread the virus while assisting high-risk patients. This study conducted routine SARS-CoV-2 screening among the staff of a specialized cardiology hospital in Brazil during 2022 and 2023, while also evaluating variables associated with infection and the occurrence of symptoms. Methods: A prospective cohort study of 94 HCWs with biweekly RT-PCR screening was performed, employing RT-PCR from nasal swabs. Results: Participants aged 50.9 ± 10.2 years and were predominantly female (85.1%) and non-white (56.4%). The follow-up period was 576.4 ± 185.9 days, and most participants worked in the intensive care unit/emergency department (34%). Although the HCWs with the highest COVID-19 rates before inclusion were technicians/graduates (67.3%) and non-white individuals (57.7%), these groups presented lower infection rates at follow-up (p < 0.001, CI 95% 2.924–27.93; and p = 0.02, CI 95% 0.129–0.859, respectively). The number of asymptomatic cases increased during the study (p = 0.001), and simultaneous infection upsurges occurred in different hospital departments. Interpretation: These data highlight the association between educational level and the risk of SARS-CoV-2 infection in HCWs. The synchronicity of cases in different hospital departments offers insights about the nosocomial spread of SARS-CoV-2. The increase in the number of asymptomatic infections with repeated infections suggests that regular molecular screening may contribute to increasing the safety of both patients and HCWs in a pandemic context.
Journal Article
Modelling the effect of defects and cracks in solar cells’ performance using the d1MxP discrete model
Renewable energies are increasingly playing an important role in the world’s energy supply. Society demands new solutions to solve environmental issues caused by fossil fuels. The importance of photovoltaic technology has been increasing and consequently, the necessity to have more accurate models to characterise the performance of solar cells during their entire lifetime has rose as well. Performance problems may appear during devices’ lifetimes associated with factors, such as weather conditions or faulty installation. Cracking might occur, leading to abrupt reductions on the produced power, quite difficult and expensive to fix. The I–V curves of a defected or cracked solar cell might not have the shape imposed by the usual models as 1M5P. In this article, cracked c-Si solar cells are modelled using a novel model: d1MxP. This model is based on the discretisation of the diode’s response on models as 1M5P. Instead of imposing a shape and compute some parameters to fit it on experimental data, the proposed model connects every two points. The results suggest a better fit using the proposed model in comparison with the 1M5P, not only in the original curves, but also modelling cracked cells. As consequence of a better fitting, the computation of important figures of merit as maximum power point or fill factor, reveals to be more precise. It is concluded that the proposed model might characterise the performance of a solar cell, even cracked, which is a huge advance aiming the possibility of simulating complex problems during the cells’ operation lifetime.
Journal Article