Adult paroxysmal cold hemoglobinuria following mRNA COVID‐19 vaccination

Abstract Paroxysmal cold hemoglobinuria (PCH) is an extremely rare subtype of autoimmune hemolytic anemia (AIHA) in adults. PCH is caused by the biphasic Donath–Landsteiner (DL) antibody which fixes complement to red blood cells at low temperatures and dissociates at warmer temperatures, leading to complement‐mediated intravascular hemolysis. Autoimmune hematological disorders including AIHA and immune thrombocytopenia have been reported to develop following the mRNA COVID‐19 vaccination. However, PCH developing subsequent to mRNA vaccination has never been reported. We report a 59‐year‐old male who developed PCH approximately a month after his second mRNA COVID‐19 vaccination

<i>Pseudomonas aeruginosa serA</i> Gene Is Required for Bacterial Translocation through Caco-2 Cell Monolayers

<div><p>To specify critical factors responsible for <i>Pseudomonas aeruginosa</i> penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to <i>P</i>. <i>aeruginosa</i> PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the <i>serA</i> mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the <i>serA</i> gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, _L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type <i>P</i>. <i>aeruginosa</i> PAO1 strain. Together, these results suggest that <i>serA</i> is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of <i>P</i>. <i>aeruginosa</i> through Caco-2 cell monolayers. Oral administration of _L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by <i>P</i>. <i>aeruginosa</i> through inhibition of <i>serA</i> function.</p></div

Influence of _L-serine addition on swarming motility in the wild-type strain.

<p>Swarming motility of the wild-type strain (WT), PAO1Tn::<i>serA</i> mutant (<i>ΔserA</i>), PAO1Tn::<i>flgE</i> mutant (<i>ΔflgE</i>) (as a negative control), and WT in the presence of _L-serine (20, 30, 40, and 50 mM). A representative image of the swarming motility assay is shown. The major axis of swarming is the longest length of the swarming area. The assay was performed in triplicate, and the results are expressed as the mean ± SD. Significant differences were observed between WT and <i>ΔserA</i> (*: P < 0.05), between WT and <i>ΔflgE</i> (*: P < 0.05), between WT and WT in the presence of 30 mM _L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM _L-serine (*: P < 0.05), and between WT and WT in the presence of 50 mM _L-serine (*: P < 0.05).</p

PCR primers used in this study.

<p>PCR primers used in this study.</p

Penetration activity of <i>P</i>. <i>aeruginosa</i> strains.

<p>(A) The wild-type strain (WT), PAO1Tn::<i>serA</i> mutant (<i>ΔserA</i>), PAO1Tn::<i>serA</i> (pUCP19-<i>serA</i>) complementary strain (+<i>serA</i>), and PAO1Tn::<i>flgE</i> mutant (<i>ΔflgE</i>) were inoculated onto the apical surfaces of Caco-2 cell monolayers at an MOI of 100, and the number of bacteria in the basolateral medium was counted at 6 h after infection. The assay was performed in triplicate, and the results are expressed as the mean ± SD. <i>E</i>. <i>coli</i> DH5α was used as a negative control. *#: P < 0.05; *: vs WT; #: vs Δ<i>serA</i> (B) Influence of _L-serine addition on penetration activity of the wild-type strain. The assay was performed in triplicate, and the results are expressed as mean ± SD. *: P < 0.05; *: vs WT.</p

Fly survival experiments.

<p>Virulence of the wild-type strain (WT), PAO1Tn::<i>serA</i> mutant (<i>ΔserA</i>), PAO1Tn::<i>serA</i> (pUCP19-<i>serA</i>) complementary strain (+<i>serA</i>), <i>E</i>. <i>coli</i> DH5α (as a negative control), and WT in the presence of _L-serine (10, 20, 30, 40, and 50 mM) was evaluated. Significant differences, based on the log-rank test, were observed between WT and DH5α (*: P < 0.05), between WT and <i>ΔserA</i> (*: P < 0.05), between WT and WT in the presence of 50 mM _L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM _L-serine (*: P < 0.05), between WT and WT in the presence of 20 mM _L-serine (*: P < 0.05), between WT and WT in the presence of 30 mM _L-serine (*: P < 0.05), and between WT and WT in the presence of 10 mM _L-serine (*: P < 0.05). Virulence of the +<i>serA</i> complementary strain was significantly restored as compared to that of <i>ΔserA</i> (#; P < 0.05).</p

Staining of bacterial flagella.

<p>Flagella staining of the wild-type strain (WT), PAO1Tn::<i>serA</i> mutant (<i>ΔserA</i>), PAO1Tn::<i>serA</i> (pUCP19-<i>serA</i>) complementary strain (+<i>serA</i>), and PAO1Tn::<i>flgE</i> mutant (<i>ΔflgE</i>) was performed as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169367#pone.0169367.ref035" target="_blank">35</a>].</p

Bacterial adherence to Caco-2 cells for the wild-type strain (WT), PAO1Tn::<i>serA</i> mutant (<i>ΔserA</i>), PAO1Tn::<i>serA</i> (pUCP19-<i>serA</i>) complementary strain (+<i>serA</i>), PAO1Tn::<i>flgE</i> mutant (<i>ΔflgE</i>), <i>E</i>. <i>coli</i> DH5α (as a negative control), and WT in the presence of _L-serine (20, 30, 40, and 50 mM).

<p>Bacterial adherence was determined based on the number of adhered bacteria per Caco-2 cell. The assay was performed in six replicates, and the results are expressed as the mean ± SD. Significant differences were observed between WT and <i>ΔserA</i> (*: P < 0.05), between WT and DH5α (*: P < 0.05), between WT and <i>ΔflgE</i> (*: P < 0.05), between WT and WT in the presence of 30 mM _L-serine (*: P < 0.05), between WT and WT in the presence of 40 mM _L-serine (*: P < 0.05), and between WT and WT in the presence of 50 mM _L-serine (*: P < 0.05). Bacterial adherence of the +<i>serA</i> complementary strain was significantly restored as compared to that of <i>ΔserA</i> (#; P < 0.05).</p

ExoS secretion assay.

<p>(A) Western blot analysis to detect secretion of ExoS into the culture supernatant using type III secretion system-inducing conditions in the wild-type strain (WT), PAO1Tn::<i>serA</i> mutant (<i>ΔserA</i>), PAO1Tn::<i>serA</i> (pUCP19-<i>serA</i>) complementary strain (+<i>serA</i>), and WT in the presence of _L-serine (30, 40, and 50 mM). A representative western blot image is shown. The arrow indicates the presence of ExoS with a deduced molecular weight of 48 kDa. (B) Expression ratio of ExoS based on western blot analyses of WT, <i>ΔserA</i>, +<i>serA</i>, and WT in the presence of 50 mM _L-serine. Western blotting was repeated three times, and bands were quantified by ImageJ. Data is shown as the ratio of ExoS expression to that of the wild-type strain and is expressed as the mean ± SD. A significant difference was observed for ExoS expression between WT and <i>ΔserA</i> (*: P < 0.05).</p