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. 2024 Sep 4:15:1449003.
doi: 10.3389/fimmu.2024.1449003. eCollection 2024.

Role of factor H-related protein 3 in Pseudomonas aeruginosa bloodstream infections

Alex González-Alsina  1 , Héctor Martín-Merinero  2 , Margalida Mateu-Borrás  1 , María Verd  1 , Antonio Doménech-Sánchez  1 , Joanna B Goldberg  3 , Santiago Rodríguez de Córdoba  2 , Sebastián Albertí  1
Affiliations

Role of factor H-related protein 3 in Pseudomonas aeruginosa bloodstream infections

Alex González-Alsina et al. Front Immunol. .

Abstract

Pseudomonas aeruginosa is a leading cause of nosocomial bloodstream infections. The outcome of these infections depends on the virulence of the microorganism as well as host-related conditions and factors. The complement system plays a crucial role in defense against bloodstream infections. P. aeruginosa counteracts complement attack by recruiting Factor H (FH) that inhibits complement amplification on the bacterial surface. Factor H-related proteins (FHRs) are a group of plasma proteins evolutionarily related to FH that have been postulated to interfere this bacterial evasion mechanism. In this study, we demonstrate that FHR-3 competes with purified FH for binding to P. aeruginosa and identify EF-Tu as a common bacterial _target for both complement regulator factors. Importantly, elevated levels of FHR-3 in human serum promote complement activation, leading to increased opsonization and killing of P. aeruginosa. Conversely, physiological concentrations of FHR-3 have no significant effect. Our findings suggest that FHR-3 may serve as a protective host factor against P. aeruginosa infections.

Keywords: FHR-3; P. aeruginosa; bloodstream infection; complement system; factor H.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
FHR-3 competes with FH for the binding to P. aeruginosa. ELISA binding assays of purified FH to the reference strain PAO1 and the serum-sensitive isogenic LPS-deficient strain PAO1Δwzz2 in the presence of FHR-3. In (A) the strain PAO1 was incubated with purified FH (20 µg/ml) in the presence of decreasing amounts of recombinant FHR-3 (x-axis on a logarithmic scale). In (B) the strains were incubated with purified FH (20 µg/ml) in the absence (black columns) or presence of FHR-3 at 0.13 µg/ml (white columns) or at 5 µg/ml (grey columns). Bound FH was detected with the mAb OX-24. Bars represents the mean of at least three independent experiments done in duplicate, and SD is indicated by error bars. Statistical analyses were performed using one-way ANOVA with multiple comparisons; P values are indicated on the bars with asterisks. ****P < 0.0001.
Figure 2
Figure 2
FHR-3 binds P. aeruginosa EF-Tu at the same site as FH. (A) The binding of recombinant FHR-3 to immobilized EF-Tu was analyzed by ELISA. Bound FHR-3 was detected with a specific monoclonal antibody. Data represent three experiments done in duplicate, and SD is indicated by error bars. (B) The sequence of the synthetic peptides (Peptide ID) covering the EF-Tu protein sequence that were used to identify FHR-3 and FH binding regions are shown. The black outline shows the Peptide ID that reacted with FHR-3 and FH. (C) The reactivity of recombinant FHR-3 (black columns) or FH (grey columns) was tested against 25 synthetic biotinylated EF-Tu 20-mer overlapping peptides by ELISA. FHR-3 and FH were detected with a specific mAb, respectively. Data represent three experiments done in duplicate, and SD is indicated by error bars shown.
Figure 3
Figure 3
FHR-3 enhances the binding of FH to P. aeruginosa in human serum. ELISA binding assays of FH to the serum-resistant strain PAO1 or the isogenic LPS-deficient serum-sensitive strain PAO1Δwzz2 incubated in FHR-3-deficient serum (A) or heat-inactivated FHR-3-deficient serum (B) (both at 10%) without (black columns) or supplemented with FHR-3 at 2 µg/ml of serum (white columns) or 100 µg/ml of serum (grey columns). FH was detected with the mAb OX-24. Bars represents the mean of at least three independent experiments done in duplicate, and SD is indicated by error bars. Statistical analyses were performed using one-way ANOVA with multiple comparisons; P values are indicated on the bars. * P< 0.05, **** P <0.001.
Figure 4
Figure 4
FHR-3 increases the deposition of C3 on P. aeruginosa. FHR-3 deficient serum (25%) without (black columns) or supplemented with FHR-3 at 2 µg/ml of serum (white columns) or at 100 µg/ml of serum (grey columns) were incubated for 15 min at 37°C with PAO1 or the isogenic serum-sensitive strain PAO1Δwzz2. Deposition of C3 on the bacterial surface was determined by ELISA. Bars represents the mean of at least three independent experiments done in duplicate, and SD is indicated by error bars. Statistical analyses were performed using one-way ANOVA with multiple comparisons; P values are indicated on the bars. ** P< 0.01.
Figure 5
Figure 5
P. aeruginosa-induced generation of C3a and C5a is enhanced by FHR-3. FHR-3 deficient serum (25%) without (black columns) or supplemented with FHR-3 at 2 µg/ml of serum (white columns) or at 100 µg/ml of serum (grey columns) were incubated for 15 min at 37°C with the serum-resistant strain PAO1 or the isogenic serum-sensitive strain PAO1Δwzz2. Levels of C3a (A) and C5a (B) were determined by ELISA. Bars represents the mean of at least three independent experiments done in duplicate, and SD is indicated by error bars. Statistical analyses were performed using one-way ANOVA with multiple comparisons; P values are indicated on the bars. ** P<0.01, *** P<0.001.
Figure 6
Figure 6
FHR-3 increases P. aeruginosa complement-mediated killing. Survival of strains PAO1 (A), PAO1Δwzz2 (B), and the clinical isolates from bloodstream infections B75 (C) and B205 (D) at different time points after incubation in strain-optimized concentrations of FHR-3 deficient serum (PAO1, 45%; PAO1Δwzz2, 2.5%; B75, 2.5% and B205; 45%) not supplemented with FHR-3 (black circles) or supplemented with FHR-3 at 2 µg/ml of serum (white circles), or 100 µg/ml of serum (grey circles). Black and grey circles are overlaid in panel A, while black and white circles are overlaid in panels C and (D) Percentage survival at each time point was calculated in respect to the number of viable bacteria at time 0. Dots represent the mean of at least three independent experiments done in duplicate, and SD is indicated by error bars. Statistical analyses were performed using one-way ANOVA with multiple comparisons. P values; * P< 0.05, ** P <0.01.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by grant PID2021-127281OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF EU “A way of making Europe”. AG-A is the recipient of FPI fellowship from Comunitat Autonoma de les Illes Balears (CAIB).

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