Thioredoxin 80-Activated-Monocytes (TAMs) Inhibit the Replication of Intracellular Pathogens

BACKGROUND: Thioredoxin 80 (Trx80) is an 80 amino acid natural cleavage product of Trx, produced primarily by monocytes. Trx80 induces differentiation of human monocytes into a novel cell type, named Trx80-activated-monocytes (TAMs). PRINCIPAL FINDINGS: In this investigation we present evidence for a role of TAMs in the control of intracellular bacterial infections. As model pathogens we have chosen Listeria monocytogenes and Brucella abortus which replicate in the cytosol and the endoplasmic reticulum respectively. Our data indicate that TAMs efficiently inhibit intracellular growth of both L. monocytogenes and B. abortus. Further analysis shows that Trx80 activation prevents the escape of GFP-tagged L. monocytogenes into the cytosol, and induces accumulation of the bacteria within the lysosomes. Inhibition of the lysosomal activity by chloroquine treatment resulted in higher replication of bacteria in TAMs compared to that observed in control cells 24 h post-infection, indicating that TAMs kill bacteria by preventing their escape from the endosomal compartments, which progress into a highly degradative phagolysosome. SIGNIFICANCE: Our results show that Trx80 potentiates the bactericidal activities of professional phagocytes, and contributes to the first line of defense against intracellular bacteria

Requirement of norD for Brucella suis Virulence in a Murine Model of In Vitro and In Vivo Infection

A mutant of Brucella suis bearing a Tn5 insertion in norD, the last gene of the operon norEFCBQD, encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. The norD strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that norD is essential for Brucella virulence

In vitro activity of omadacycline, a new tetracycline analog, and comparators against Clostridioides difficile.

Omadacycline is a potent aminomethylcycline with in vitro activity against Gram-positive, Gram-negative, and anaerobic bacteria. Preliminary data demonstrated that omadacycline has in vitro activity against Clostridioides difficile; however, large-scale in vitro studies have not been done. The purpose of this study was to assess the in vitro susceptibility of omadacycline and comparators on a large biobank of clinical C. difficile isolates. In vitro C. difficile susceptibility to omadacycline and comparators (fidaxomicin, metronidazole, and vancomycin) was assessed using the broth microdilution method. Minimum bactericidal concentrations (MBCs) and time-kill assays were assessed for pharmacodynamics analysis, and whole-genome sequencing was performed in a subset of isolates to assess distribution of MICs and resistance determinants. Two hundred fifty clinical C. difficile isolates collected between 2015 and 2018 were tested for in vitro susceptibility of omadacycline and comparators. Ribotypes included F001 (n = 5), F002 (n = 56), F014-020 (n = 66), F017 (n = 8), F027 (n = 53), F106 (n = 45), and F255 (n = 17). Omadacycline demonstrated potent in vitro activity with an MIC range of 0.016 to 0.13 μg/ml, an MIC50 of 0.031 μg/ml, and an MIC90 of 0.031 μg/ml. No difference was observed for omadacycline MIC50 and MIC90 values stratified by ribotype, disease severity, or vancomycin susceptibility. Bactericidal activity was confirmed in time-kill studies. No difference was observed in MIC based on C. difficile phylogeny. Further development of omadacycline as an intravenous and oral antibiotic directed toward C. difficile infection is warranted

Growth curve of untreated control monocytes and TAMs after <i>L. monocytogenes</i> infection.

<p>Cells were infected as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. After the infection, samples were taken at the indicated periods of time, and number of viable cells was assayed by trypan blue exclusion. The data are presented as number of viable cells (panel A) and as percentage of cell recovery, being 100% of recovery at time 0 (panel B). Mean ± SEM of six independent experiments.</p

TAMs control replication of <i>Listeria monocytogenes.</i>

<p><b>A</b>. Intracellular growth curve of <i>L. monocytogenes</i>. Cells were infected for 30 minutes at 37°C with a multiplicity of infection of 25∶1 bacteria per cell, and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and RPMI supplemented with 5 µg/ml gentamicin was added. At the indicated periods of time, 3×10⁵ cells were lysed and colony forming units CFU were quantified, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. A representative experiment is shown. <b>B</b>. Summary of the growth curves of <i>L. monocytogenes</i>. The data are presented as percentage of the number of CFU recovered in TAMs relative to the number of CFU recovered in control monocytes (mean ± SEM of five independent experiments). <b>C</b>. Cells were infected with <i>L. monocytogenes</i> for 30 minutes at 37°C with a MOI 25∶1 and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria. Intracellular bacteria were detected by immunofluorescence analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a> (mean ± SEM of five independent experiments).</p

Localization of GFP-tagged <i>L. monocytogenes</i> in acidic compartments.

<p><b>A</b>. Cells were infected with the GFP-tagged <i>L. monocytogenes</i> at MOI 50∶1 for 4 hours, and treated with the acidotropic dye Lysotracker Red. Cytospin centrifugation was performed and cells were fixed with 4% paraformaldehyde. Lysotracker Red positive vesicles containing GFP-labeled bacteria are indicated with arrows. Bar represents 5 µm. <b>B</b>. The percentage of Lysotracker Red positive vesicles containing GFP-labeled bacteria was quantified in two independent experiments. A minimum of 200 cells were analyzed. <b>C</b>. Scatter plot analysis showing the correlation between the GFP and Lysotracker Red fluorescence intensities in control cells and TAMs quantified by the Volocity software. A correlation coefficient value of r² equal to 1 indicates 100% bacterial localization with the lysosomal compartments.</p

Escape of <i>L. monocytogenes</i> NF-L327 into the cytosol of infected monocytes.

<p><b>A</b>. Cells were infected with the <i>L. monocytogenes</i> strain NF-L327, that expresses GFP when translocated into the cytosol, at MOI 50∶1 for 30 minutes and medium containing 100 µg/ml gentamicin was added for 1 h to kill extracellular bacteria (0 h). Cells were washed and maintained in RPMI supplemented with 5 µg/ml gentamicin. Cells were lysed after infection (0 h) and 24 h in culture and the colony forming units CFU were quantified, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#pone-0016960-g003" target="_blank">Figure 3A</a>. A representative experiment is shown. <b>B</b>. Cells were infected with the <i>L. monocytogenes</i> NF-L327 at MOI 50∶1 as described in A and samples were taken 8 hours post-infection. Cytospin and fluorescence imaging were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016960#s2" target="_blank">Materials and Methods</a>. Bar represents 5 µm. <b>C</b>. Quantification of GFP-labeled <i>Listeria</i> was performed using the Volocity software. The data are presented as number of bacteria in 200 cells (SEM of two independent experiments).</p