Structural insights into VirB-DNA complexes reveal mechanism of transcriptional activation of virulence genes

VirB activates transcription of virulence genes in Shigella flexneri by alleviating heat-stable nucleoid-structuring protein-mediated promoter repression. VirB is unrelated to the conventional transcriptional regulators, but homologous to the plasmid partitioning proteins. We determined the crystal structures of VirB HTH domain bound by the cis-acting site containing the inverted repeat, revealing that the VirB-DNA complex is related to ParB-ParS-like complexes, presenting an example that a ParB-like protein acts exclusively in transcriptional regulation. The HTH domain of VirB docks DNA major groove and provides multiple contacts to backbone and bases, in which the only specific base readout is mediated by R167. VirB only recognizes one half site of the inverted repeats containing the most matches to the consensus for VirB binding. The binding of VirB induces DNA conformational changes and introduces a bend at an invariant A-tract segment in the cis-acting site, suggesting a role of DNA remodeling. VirB exhibits positive cooperativity in DNA binding that is contributed by the C-terminal domain facilitating VirB oligomerization. The isolated HTH domain only confers partial DNA specificity. Additional determinants for sequence specificity may reside in N- or C-terminal domains. Collectively, our findings support and extend a previously proposed model for relieving heat-stable nucleoid-structuring protein-mediated repression by Vir

Structural Insight Into Conformational Changes Induced by ATP Binding in a Type III Secretion-Associated ATPase From Shigella flexneri

Gram-negative bacteria utilize the type III secretion system (T3SS) to inject effector proteins into the host cell cytoplasm, where they subvert cellular functions and assist pathogen invasion. The conserved type III-associated ATPase is critical for the separation of chaperones from effector proteins, the unfolding of effector proteins and translocating them through the narrow channel of the secretion apparatus. However, how ATP hydrolysis is coupled to the mechanical work of the enzyme remains elusive. Herein, we present a complete description of nucleoside triphosphate binding by surface presentation antigens 47 (Spa47) from Shigella flexneri, based on crystal structures containing ATPγS, a catalytic magnesium ion and an ordered water molecule. Combining the crystal structures of Spa47-ATPγS and unliganded Spa47, we propose conformational changes in Spa47 associated with ATP binding, the binding of ATP induces a conformational change of a highly conserved luminal loop, facilitating ATP hydrolysis by the Spa47 ATPase. Additionally, we identified a specific hydrogen bond critical for ATP recognition and demonstrated that, while ATPγS is an ideal analog for probing ATP binding, AMPPNP is a poor ATP mimic. Our findings provide structural insight pertinent for inhibitor design

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Structural insights into VirB-DNA complexes reveal mechanism of transcriptional activation of virulence genes

VirB activates transcription of virulence genes in Shigella flexneri by alleviating heat-stable nucleoid-structuring protein-mediated promoter repression. VirB is unrelated to the conventional transcriptional regulators, but homologous to the plasmid partitioning proteins. We determined the crystal structures of VirB HTH domain bound by the cis-acting site containing the inverted repeat, revealing that the VirB-DNA complex is related to ParB-ParS-like complexes, presenting an example that a ParB-like protein acts exclusively in transcriptional regulation. The HTH domain of VirB docks DNA major groove and provides multiple contacts to backbone and bases, in which the only specific base readout is mediated by R167. VirB only recognizes one half site of the inverted repeats containing the most matches to the consensus for VirB binding. The binding of VirB induces DNA conformational changes and introduces a bend at an invariant A-tract segment in the cis-acting site, suggesting a role of DNA remodeling. VirB exhibits positive cooperativity in DNA binding that is contributed by the C-terminal domain facilitating VirB oligomerization. The isolated HTH domain only confers partial DNA specificity. Additional determinants for sequence specificity may reside in N- or C-terminal domains. Collectively, our findings support and extend a previously proposed model for relieving heat-stable nucleoid-structuring protein-mediated repression by Vir

Structure-Based Prototype Peptides Targeting the Pseudomonas aeruginosa Type VI Secretion System Effector as a Novel Antibacterial Strategy

The type VI secretion system (T6SS) secretes numerous toxins for bacteria-bacteria competition. TplE is a newly identified trans-kingdom toxin secreted by the T6SS in Pseudomonas aeruginosa, while TplEi neutralizes the toxic effect of TplE to protect bacteria autointoxication. Blocking the interaction of TplE-TplEi could unleash the toxin, causing bacterial cell death. In this study, we applied a crystallographic approach to design a structural-based antimicrobial peptides targeting the interaction of TplE and TplEi. We found that a peptide (designed as “L” peptide based on its shape) derived from TplE can form a crystal complex with TplEi after subtilisin treatment and the crystal structure was solved at 2.2Å. The “L” peptide displays strong binding affinity to TplEi in vitro and can release the TplE toxin to induce bacteria death in vivo. Our findings suggest that as a toxin activator, the “L” peptide could be a possible drug lead for treating P. aeruginosa infection. Our findings provide an example that the T6SS effector and immunity protein could be a potential drug target against bacteria infection

Stabilization of the Metastable Pre-Fusion Conformation of the SARS-CoV-2 Spike Glycoprotein through N-Linked Glycosylation of the S2 Subunit

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the novel coronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic, represents a serious threat to public health. The spike (S) glycoprotein of SARS-CoV-2 mediates viral entry into host cells and is heavily glycosylated. In this study, we systemically analyzed the roles of 22 putative N-linked glycans in SARS-CoV-2 S protein expression, membrane fusion, viral entry, and stability. Using the α-glycosidase inhibitors castanospermine and NB-DNJ, we confirmed that disruption of N-linked glycosylation blocked the maturation of the S protein, leading to the impairment of S protein-mediated membrane fusion. Single-amino-acid substitution of each of the 22 N-linked glycosylation sites with glutamine revealed that 9 out of the 22 N-linked glycosylation sites were critical for S protein folding and maturation. Thus, substitution at these sites resulted in reduced S protein-mediated cell–cell fusion and viral entry. Notably, the N1074Q mutation markedly affected S protein stability and induced significant receptor-independent syncytium (RIS) formation in HEK293T/hACE2-KO cells. Additionally, the removal of the furin cleavage site partially compensated for the instability induced by the N1074Q mutation. Although the corresponding mutation in the SARS-CoV S protein (N1056Q) did not induce RIS in HEK293T cells, the N669Q and N1080Q mutants exhibited increased fusogenic activity and did induce syncytium formation in HEK293T cells. Therefore, N-glycans on the SARS-CoV and SARS-CoV-2 S2 subunits are highly important for maintaining the pre-fusion state of the S protein. This study revealed the critical roles of N-glycans in S protein maturation and stability, information that has implications for the design of vaccines and antiviral strategies

Structural insights into VirB-DNA complexes reveal mechanism of transcriptional activation of virulence genes

ISSN:1362-4962ISSN:0301-561