Iron Acquisition Proteins of Pseudomonas aeruginosa as Potential Vaccine Targets: In Silico Analysis and In Vivo Evaluation of Protective Efficacy of the Hemophore HasAp

Abstract

Background: Pseudomonas aeruginosa (PA) is a Gram-negative pathogen responsible for fatal nosocomial infections worldwide. Iron is essential for Gram-negative bacteria to establish an infection. Therefore, iron acquisition proteins (IAPs) of bacteria are attractive vaccine targets. Methodology: A “Reverse Vaccinology” approach was employed in the current study. Expression levels of 37 IAPs in various types of PA infections were analyzed in seven previously published studies. The IAP vaccine candidate was selected based on multiple criteria, including a high level of expression, high antigenicity, solubility, and conservation among PA strains, utilizing suitable bioinformatics analysis tools. The selected IAP candidate was recombinantly expressed in Escherichia coli and purified using metal affinity chromatography. It was further evaluated in vivo for protection efficacy. The novel immune adjuvant, naloxone (NAL), was used. Results and discussion: HasAp antigen met all the in silico selection criteria, being highly antigenic, soluble, and conserved. In addition, it was the most highly expressed IAP in terms of average fold change compared to control. Although HasAp did excel in the in silico evaluation, subcutaneous immunization with recombinant HasAp alone or recombinant HasAp plus NAL (HasAP-NAL) did not provide the expected protection compared to controls. Immunized mice showed a low IgG2a/IgG1 ratio, indicating a T-helper type 2 (Th2)-oriented immune response that is suboptimal for protection against PA infections. Surprisingly, the bacterial count in livers of both NAL- and HasAp-NAL-immunized mice was significantly lower than the count in the HasAp and saline groups. The same trend was observed in kidneys and lungs obtained from these groups, although the difference was not significant. Such protection could be attributed to the enhancement of innate immunity by NAL. Conclusions: We provided a detailed in silico analysis of IAPs of PA followed by in vivo evaluation of the best IAP, HasAp. Despite the promising in silico results, HasAp did not provide the anticipated vaccine efficacy. HasAp should be further evaluated as a vaccine candidate through varying the immunization regimens, models of infection, and immunoadjuvants. Combination with other IAPs might also improve vaccination efficacy. We also shed light on several highly expressed promising IAPs whose efficacy as vaccine candidates is worthy of further investigation.

Integration of immunoinformatics and cheminformatics to design and evaluate a multitope vaccine against Klebsiella pneumoniae and Pseudomonas aeruginosa coinfection

“…These proteins have been selected as targets for vaccine design against several microorganisms including the current study targeted bacteria (Farhadi et al, 2015;Jahangiri et al, 2017;Dey et al, 2022). In addition to that, bacterial iron uptake proteins have been also selected in several trials as promising candidates for vaccine development against K. pneumoniea and P. aeruginosa (Nemati Zargaran et al, 2021;Hamad et al, 2023). Iron is an essential micronutrient for most bacteria that is used for many essential cellular processes where it is obtained from iron-chelating siderophores or directly from iron-containing host proteins.…”

Exploring aggregation genes in aP. aeruginosachronic infection model

“…This pipeline uses sequential and structural homology to assign gene functions and co-expression patterns to predict protein participation within known pathways. Interestingly, only three genes have known or suggested links to previously reported Pa biofilm functions: PA3406 (hasD) a component of the HasAD hemophore, PA0998 (pqsC) a component of the PQS quorum sensing (QS) pathway, and hypothetical protein PA1864 which has homology to a transcriptional repressor of FimWX mediated surface adhesion (Table 1) (28)(29)(30)(31). PA1864 and hasD were upregulated in aggregate conditions 27.98 and 39.02-fold, respectively, while pqsC was downregulated -7.25-fold.…”

Improving Protection to Prevent Bacterial Infections: Preliminary Applications of Reverse Vaccinology against the Main Cystic Fibrosis Pathogens

“…PA is classified as multi-drug resistant (MDR), characterized by a number of virulence factors and an adaptable genome, which will make the microorganism able to overlap the ongoing treatments. For this reason, the research for new antibiotics and antivirulence compounds is pursued simultaneously with that for vaccines against P. aeruginosa [ 65 , 66 , 67 ]. Vaccine targets include factors that trigger the immune response in the host, characterized by antigenic elements typically found on the bacterial surface, such as lipopolysaccharides (LPS) and outer membrane proteins (OMPs) [ 68 ].…”

“…Since iron is essential for Gram-negative bacteria to establish infections, iron acquisition proteins (IAPs) are attractive targets for vaccine development, including against P. aeruginosa [ 65 ]. Bioinformatics analysis allowed us to choose the best target, taking into account many features such as frequency of expression, antigenicity and solubility, which are important for the subsequent building of recombinant proteins.…”

“…The development of a multi-epitope vaccine can start from these assumptions, merging many epitopes from different IAPs. For instance, HasAP, PhuR and HxuA, which are involved in the iron scavenging from porphyrins have been evaluated as the target for the development of new vaccines [ 65 ].…”