Participation of the <i>Salmonella</i> OmpD Porin in the Infection of RAW264.7 Macrophages and BALB/c Mice

<div><p><i>Salmonella</i> Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, <i>Salmonella</i> must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the <i>Salmonella</i> Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a Δ<i>ompD</i> strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of <i>ompD</i> caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the Δ<i>ompD</i> strain showed increased ability to survive and replicate in target organs of infection. The <i>ompD</i> transcript levels showed a down-regulation when <i>Salmonella</i> resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the Δ<i>ompD</i> strain produced lower levels of reactive oxygen species, suggesting that down-regulation of <i>ompD</i> could favor replication of <i>Salmonella</i> inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host.</p></div

Expression of <i>S.</i> Typhimurium 14028 s <i>ompD</i> inside macrophages.

<p>Transcript levels of <i>ompD</i> (A) and <i>ompW</i> (B) from strain 14028 s were quantified by qRT-PCR by recovering total RNA from the infected macrophages at 1 to 12 h post infection, and from bacteria of the inoculating culture (I). Experiments were performed in triplicate. Asterisks represent significant statistical differences between the control and mutants strains (* <i>p</i>≤0.05; ** <i>p</i>≤0.005;*** <i>p</i>≤0.001). Values are mean ± SD.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Effect of OmpD in systemic infection.

<p>(A) Bacteremia assays. Groups of six BALB/c mice were orally inoculated with 10³ bacteria of strains 14028 s, Δ<i>ompD</i> or Δ<i>ompW</i> (n = 6). CFU/ml were determined from samples of peripheral blood obtained after 3 and 5 days post infection. Values are mean ± SD. Asterisks represent statistical significant differences (T-test, * <i>p</i>≤0.05). (B to G) CI assays. A mixture (1∶1 ratio) of 10³ bacteria of strains 14028 s (WT) and Δ<i>ompD</i> (B and E), Δ<i>ompW</i> (C and F) or Δ<i>ssrB</i> (D and G) was orally (B, C and D) or intraperitoneally (E, F and G) inoculated to groups of six BALBc mice (n = 6). Liver and spleen were extracted from mice 5 days post infection and the CFU per g of organ was determined for the wild-type strain and the respective mutant. Each data point represents the competitive index (CI), which is the ratio of mutant/WT of recovered CFU/g tissue in the respective organ from each mouse. Horizontal bars represent the median values of ratios for the organ type.</p

Effect of OmpD in the proliferation of <i>S</i>. Typhimurium inside macrophages.

<p>Murine RAW 264.7 macrophages were infected (MOI of 100∶1) with <i>S</i>. Typhimurium 14028 s, and its Δ<i>ompD</i>, Δ<i>ompW</i> and Δ<i>ssrB</i> derivative mutants, containing or not the pBAD vector, or the plasmids pBAD-<i>ompD</i> or pBAD-<i>ompW</i> that overexpress OmpD and OmpW, respectively. CFU of the different strains were determined after recovery from infected macrophages at the indicated time points. The relative percentage of invasion at 2 h post infection (A) and of proliferation at 6, 8 and 12 h post infection (B) was determined to analyze the effect of the absence of <i>ompD</i>, <i>ompW</i> or <i>ssrB</i> genes. The relative percentage of proliferation at 6 (C), 8 (D) and 12 h (E) post infection was determined to analyze the effect of the absence and overexpression of OmpD. The relative percentage of proliferation at 12 h (F) post infection was determined to analyze the effect of the absence and overexpression of OmpW. Asterisks represent significant statistical difference between 14028 s and mutants used in this study (* <i>p</i>≤0.05). Values are mean ± SD.</p

Intracellular levels of total ROS in RAW 264.7 macrophages infected and similarity between OmpD and other members of the OMP superfamily.

<p>(A) Total ROS were determined at 1 to 12 h post infection in macrophages infected with strains 14028 s and Δ<i>ompD</i> using the oxidant-sensitive probe H2DCFDA (fluorescence adjusted per µg of total protein of the sample). Asterisks represent statistical significant differences (* p≤0.05). A.U.  =  arbitrary units. (B) Sequence similarity network of OmpD and its closest homologues in the Omp superfamily. Nodes represent protein sequences, and edges represent worst reciprocal blastp E-values that are higher than a given threshold. Visualization was performed using the organic layout in Cytoscape 2.8.3 [Cline et al. 2007]. Reviewed proteins from Uniprot that have evidence of existing at the protein level are shown in color. Squares correspond to proteins that have known crystal structures in the Protein Data Bank. Edges filtered to e-value <1e-14, median alignment length: 341 residues, median identity: 34.0%. (C) Electrostatic surface potential of the biological assemblies of OmpD and SLAM-recognized proteins OmpC and OmpF. c.1) Periplasmic loops are mapped into the molecular surface: L1 blue, L2 red, L3 violet, L4 orange, L5 yellow, L6 ochre, L7 green and L8 Pink; c.2) Calculated surface electrostatic potential of OmpF from <i>Salmonella</i> Typhi (PDB: 3nsg); OmpF from <i>E. coli</i> (PDB:2zfg); OmpC from <i>Salmonella</i> Typhi (PDB: 3uu2) and OmpC from <i>E. coli</i> (PDB:2j1n) contoured at +- 2.0 kT.</p

<i>ompD</i> expression in target organs from BALB/c mice infected with <i>S.</i> Typhimurium 14028 s.

<p>Bacteria were recovered from organs (liver and spleen) after three and five days post infection. The <i>ompD</i> (A) and <i>ompW</i> (B) transcript levels were measured by qRT-PCR and are indicated as relative transcript levels with respect to the levels of each gene in the bacterial inoculum (I) used for infecting mice (n = 3). Experiments were performed in biological and technical triplicate. Asterisks represent statistical differences between control and treated cells (* <i>p</i>≤0.05; *** <i>p</i>≤0.001). Values are mean ± SD.</p

Global attitudes in the management of acute appendicitis during COVID-19 pandemic: ACIE Appy Study

Background: Surgical strategies are being adapted to face the COVID-19 pandemic. Recommendations on the management of acute appendicitis have been based on expert opinion, but very little evidence is available. This study addressed that dearth with a snapshot of worldwide approaches to appendicitis. Methods: The Association of Italian Surgeons in Europe designed an online survey to assess the current attitude of surgeons globally regarding the management of patients with acute appendicitis during the pandemic. Questions were divided into baseline information, hospital organization and screening, personal protective equipment, management and surgical approach, and patient presentation before versus during the pandemic. Results: Of 744 answers, 709 (from 66 countries) were complete and were included in the analysis. Most hospitals were treating both patients with and those without COVID. There was variation in screening indications and modality used, with chest X-ray plus molecular testing (PCR) being the commonest (19\ub78 per cent). Conservative management of complicated and uncomplicated appendicitis was used by 6\ub76 and 2\ub74 per cent respectively before, but 23\ub77 and 5\ub73 per cent, during the pandemic (both P < 0\ub7001). One-third changed their approach from laparoscopic to open surgery owing to the popular (but evidence-lacking) advice from expert groups during the initial phase of the pandemic. No agreement on how to filter surgical smoke plume during laparoscopy was identified. There was an overall reduction in the number of patients admitted with appendicitis and one-third felt that patients who did present had more severe appendicitis than they usually observe. Conclusion: Conservative management of mild appendicitis has been possible during the pandemic. The fact that some surgeons switched to open appendicectomy may reflect the poor guidelines that emanated in the early phase of SARS-CoV-2