AliC and AliD of nonencapsulated Streptococcus pneumoniae enhance virulence in a Galleria mellonella model of infection by contributing to reactive oxygen species resistance

Abstract

Introduction: Nonencapsulated Streptococcus pneumoniae (NESp) are isolated worldwide. Due to the lack of capsule in NESp strains the current vaccines, that target the pneumococcal capsule are ineffective. Some NESp contain the oligopeptide transporters AliC and AliD which are required for virulence through unknown mechanisms. AliC and AliD have been previously shown to reduce rates of phagocytosis and alter the transcriptome and proteome of MNZ41. We hypothesize that oligopeptide regulated genes are responsible for reduced phagocytosis and increased survival through resistance to reactive oxygen species (ROS).

Methods: To test this a mutant library of AliC and AliD regulated genes was used in in vitro and in vivo models. ROS resistance was tested through quantifying bacterial counts after exposure to hydrogen peroxide (H₂O₂). A modified surface killing assay was also used to calculate resistance to phagocytosis of our mutant library. A Galleria mellonella larvae model of infection was used to determine survival curve analyses.

Results: Two mutant genes in our library, ∆lytFN1 (CDT04) and ∆mgtC (CDT05), displayed greater sensitivity to H₂O₂ killing and phagocytosis compared to wildtype MNZ41. Deletion of AliD in an AliD-expressing encapsulated strain reduced virulence.

Conclusion: This research demonstrates that proteins encoded by genes regulated by AliC and AliD alter susceptibility to host-derived mechanisms for bacterial clearance and increases bacterial survival in response to ROS.

Keywords: Galleria mellonella; Streptococcus pneumoniae; host-pathogen; oligopeptide transporter; virulence factor.

Figure 1

Gene selection and identification of virulence genes. This figure displays a flowchart demonstrating the selection process that led to the identification of virulence genes lytFN1 and mgtC. The 2,185 genes of MNZ41 were screened using proteomics and transcriptomics. Analysis of the ‘omics data determined that 42 of those genes were regulated by oligopeptide binding protein AliD. Out of the 42 AliD-regulated genes, 7 mutants were created and used in in vivo screening, which determined that the 2 genes lytFN1 and mgtC were important in virulence. Figure created using BioRender (https://biorender.com/).

Figure 2

Kaplan-Meier analysis of survival in the Galleria mellonella model infected with wildtype pneumococci and respective mutants. Galleria mellonella larvae were infected with each pneumococcal strain, and survival was monitored for 72 hours. Kaplan-Meier survival curves were determined for WT NESp (MNZ41) and isogenic mutants JLB01 (MNZ41ΔaliCΔaliD), CDT04 (MNZ41ΔlytFN1), and CDT05 (MNZ41ΔmgtC) at a concentration of 5×10⁸ CFU/mL (A). Kaplan-Meier survival curves were also determined for WT encapsulated serotype 38 SPJV40 and its isogenic mutant CDT11 (SPJV40ΔaliD) at a concentration of 1.85×10⁷ CFU/mL (B). Data from at least three biological replicates, n=10 larvae per replicate, were used to calculate median values for graphs. Survival curves were analyzed for statistical significance using the log-rank test. A P value of <0.05 was considered to be statistically significant.

Figure 3

Murine colonization and infection with wildtype and lytFN1 mutant. Mice were infected intranasally with either wildtype MNZ41 or the isogenic lytFN1 mutant (CDT04) in an aspiration pneumonia model. After 48 hours, mice were euthanized and tissues collected for bacterial enumeration. Significantly fewer bacteria were collected from the nasopharynx when infected with CDT04 compared to MNZ41 (A). Infection with either MNZ41 or CDT04 demonstrated no differences in the number of pneumococci collected from either the lungs (B) or the middle ear (C). Data was collected from two independent infections. Error bars represent the standard errors of the means.

Figure 4

Pneumococci gene regulation. AliC/AliD regulation of lytFN1 and mgtC in NESp and AliD regulation of mgtC in an encapsulated serotype 38 strain were analyzed by RT-qPCR. Total RNA was extracted from MNZ41 (NESp) and JLB01 (MNZ41ΔaliCΔaliD) (A, B) and from SPJV40 (serotype 38) and CDT11 (SPJV40ΔaliD) (C). cDNA was generated and utilized as a template in RT-qPCRs with primers that amplified the lytFN1 and mgtC genes, respectively. Total number of lytFN1 transcripts were normalized to the gyrA transcript numbers and presented as total number of transcripts when above limit of detection. Average C_T values were normalized to the value for the gyrA gene. The fold differences were calculated using the comparative C_T (2^-ΔΔC _T) method. Panels show data from two independent biological replicates. Error bars represent the standard errors of the means.

Figure 5

Modified surface killing assay. Pneumococcal strains were exposed to differentiated HL-60 cells at a ratio of 1:100, and phagocytosis survival percentages were calculated. The percentages of survival after phagocytosis of strains MNZ41 (WT NESp), JLB01 (MNZ41ΔaliCΔaliD), CDT04 (MNZ41ΔlytFN1), and CDT05 (MNZ41ΔmgtC) were calculated by comparing the CFU of strains incubated with and those incubated without neutrophils (A). The percentages of survival after phagocytosis of strains SPJV40 (serotype 38) and CDT11 (SPJV40ΔaliD) were calculated by comparing the CFU of strains incubated with and those incubated without neutrophils (B). The percentages of survival after phagocytosis of strains R36A (unencapsulated laboratory strain) and CDT08 (R36A;pABG5::aliD) were calculated by comparing the CFU of strains incubated with and those incubated without neutrophils (C). Bar graphs are representative of results from three independent experiments performed in triplicate. Error bars represent the standard errors of the means.

Figure 6

H₂O₂ resistance. Pneumococcal strains were treated with 2.5 mM of H₂O₂ in THY or with THY only for 2 hours and serial dilutions were plated on BA. Resistance to H₂O₂ was calculated for NESp strains MNZ41 (WT), JLB01 (MNZ41 ΔaliCΔaliD), CDT04 (MNZ41ΔlytFN1), and CDT05 (MNZ41ΔmgtC) by comparing the CFU of strains incubated with and those without H₂O₂ (A). Resistance to H₂O₂ was calculated for serotype 38 strains SPJV40 (WT) and CDT11 (SPJV40ΔaliD) by comparing the CFU of strains incubated with and those without H₂O₂ (B). Resistance to H₂O₂ was calculated for unencapsulated laboratory strains R36A (WT) and CDT08 (R36A; pABG5::aliD) by comparing the CFU of strains incubated with and those without H₂O₂ (C). Experiments were performed in triplicate, and the data are shown as the mean of triplicate wells. A P value of <0.05 was considered to be statistically significant.

Figure 7

Model of mechanism. Genes lytFN1 and mgtC were identified as potential virulence factors using the Galleria mellonella infection model. In vitro experiments identified deficiencies in survival following phagocytosis when either of these genes are deleted along with susceptibility to H₂O₂ in the lytFN1 mutant. S. pneumoniae strains expressing lytFN1, which contains homology to peptidase C14 caspase, may activate apoptosis leading to neutrophil death and bacterial survival. The mgtC gene facilitates magnesium uptake, which can promote intracellular survival following phagocytosis independently of H₂O₂ mediated clearance mechanisms. Figure created using BioRender (https://biorender.com/).