Klebsiella pneumoniae Lipopolysaccharides Serotype O2afg Induce Poor Inflammatory Immune Responses Ex Vivo

Abstract

Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium.

Keywords: Klebsiella pneumoniae; NF-κB; antimicrobial resistance; immune evasion; lipopolysaccharides; nosocomial infection.

Figure 1

Following K. pneumoniae infection or colonization, titers of the antibodies against LPS O1, O2a and O2afg present in human patients’ sera were determined in infection and in colonization cases (A). Statistical analysis was performed using the Mann–Whitney U test, **** p < 0.0001. Immunoblots used different LPS quantities (1, 0.5, 0.25 and 0.125 µg) and sera from an infection case, a colonization patient and a negative individual at a dilution of 1:1000 (B).

Figure 2

The stimulation of isolated monocytes for 48 h (A–E) and 6 h (F–L) with 1 µg/mL of each of the different K. pneumoniae purified LPS (KpO1, KpO2, KpO2afg (6613) and KpO2afg (ST258)). LPS refers to E. coli 0111:B4 commercial LPS and it was also used at a concentration of 1 µg/mL. Results are representative of four different donors. The results are expressed as the average ± SEM. Statistical analysis was performed using one-way ANOVA, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 3

Whole blood cells were stimulated with 1 µg/mL of each of the K. pneumoniae purified LPS KpO1, KpO2, KpO2afg (6613) and KpO2afg (ST258) for 6 h (A–E). LPS refers to E. coli 0111:B4 commercial LPS used at the same concentration as the K. pneumoniae LPS. Results are representative of four different donors. The results are expressed as the average ± SEM. Statistical analysis was performed using one-way ANOVA, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 4

Phagocytosis of K. pneumoniae strains (KpO1, KpO2, KpO2afg (6613) and KpO2afg (ST258)) in peripheral whole blood. (A) Flow cytometry plots with gating strategy and phagocytic activity levels of (B) granulocytes and (C) monocytes are shown. Phagocytosis was measured after 60 min of incubation with stained bacteria using the pHrodo™ Red Phagocytosis Particle labelling kit. CTRL refers to the control used, i.e., phagocytosis blocked by incubation in ice for 60 min, and is representative of what was observed for all controls of the K. pneumoniae strains used in this experiments. Statistical analysis was done using ordinary one-way ANOVA, **** p < 0.0001.

Figure 5

Transcriptional levels of fimA (A), fimH (B) and ompK36 (C). K. pneumoniae mRNA from strains B5055, C5046, 6613 and St258 were quantified by Real-Time PCR. rho was used as a reference gene for the relative quantification and assessed by 2^−ΔΔCT calculation for each mRNA. Fold changes were calculated based on B5055 transcriptional levels.