The small RNA PrrH aggravates Pseudomonas aeruginosa-induced acute lung injury by regulating the type III secretion system activator ExsA

Qixuan Shi; Shenghe Zeng; Ruiqi Yu; Mo Li; Cong Shen; Xuan Zhang; Chanjing Zhao; Jianming Zeng; Bin Huang; Jieying Pu; Cha Chen

doi:10.1128/spectrum.00626-23

. 2024 Jan 30;12(3):e00626-23. doi: 10.1128/spectrum.00626-23

The small RNA PrrH aggravates Pseudomonas aeruginosa-induced acute lung injury by regulating the type III secretion system activator ExsA

Qixuan Shi ^1,^2,^#, Shenghe Zeng ^1,^2,^#, Ruiqi Yu ^1,², Mo Li ¹, Cong Shen ¹, Xuan Zhang ¹, Chanjing Zhao ¹, Jianming Zeng ¹, Bin Huang ^3,^✉, Jieying Pu ^1,^✉, Cha Chen ^1,^✉

Editor: Fernando Navarro-Garcia⁴

PMCID: PMC10913731 PMID: 38289930

ABSTRACT

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals. Small regulatory RNAs (sRNAs) regulate multiple bacterial adaptations to environmental changes, especially virulence. Our previous study showed that sRNA PrrH negatively regulates the expression of a number of virulence factors, such as pyocyanin, rhamnolipid, biofilm, and elastase in the P. aeruginosa strain PAO1. However, previous studies have shown that the prrH-deficient mutant attenuates virulence in an acute murine lung infection model. All ΔprrH-infected mice survived the entire 28-day course of the experiment, whereas all mice inoculated with the wild-type or the complemented mutant succumbed to lung infection within 4 days of injection, but the specific mechanism is unclear. Herein, we explored how PrrH mediates severe lung injury by regulating the expression of virulence factors. In vivo mouse and in vitro cellular assays demonstrated that PrrH enhanced the pathogenicity of PAO1, causing severe lung injury. Mechanistically, PrrH binds to the coding sequence region of the mRNA of exsA, which encodes the type III secretion system master regulatory protein. We further demonstrated that PrrH mediates a severe inflammatory response and exacerbates the apoptosis of A549 cells. Overall, our results revealed that PrrH positively regulates ExsA, enhances the pathogenicity of P. aeruginosa, and causes severe lung injury.

IMPORTANCE

Pseudomonas aeruginosa is a Gram-negative bacterium and the leading cause of nosocomial pneumonia. The pathogenicity of P. aeruginosa is due to the secretion of many virulence factors. Small regulatory RNAs (sRNAs) regulate various bacterial adaptations, especially virulence. Therefore, understanding the mechanism by which sRNAs regulate virulence is necessary for understanding the pathogenicity of P. aeruginosa and the treatment of the related disease. In this study, we demonstrated that PrrH enhances the pathogenicity of P. aeruginosa by binding to the coding sequence regions of the ExsA, the master regulatory protein of type III secretion system, causing severe lung injury and exacerbating the inflammatory response and apoptosis. These findings revealed that PrrH is a crucial molecule that positively regulates ExsA. Type III-positive strains are often associated with a high mortality rate in P. aeruginosa infections in clinical practice. Therefore, this discovery may provide a new target for treating P. aeruginosa infections, especially type III-positive strains.

KEYWORDS: Pseudomonas aeruginosa, PrrH, T3SS, ExsA, lung injury

INTRODUCTION

Pseudomonas aeruginosa is a multidrug-resistant opportunistic pathogen that causes acute and chronic infections in immunocompromised individuals with chronic obstructive pulmonary disease, cystic fibrosis, and serious burns. P. aeruginosa is the most common cause of nosocomial pneumonia among Gram-negative bacteria (1 –6). And in recent years, P. aeruginosa infections cause high mortality due to its high drug resistance and virulence (7 –9), which makes them extremely difficult to treat (10 –14). Therefore, understanding the underlying mechanisms of P. aeruginosa-induced pneumonia is crucial for the treatment of the infection.

P. aeruginosa can adapt to the host environment due to its many virulence factors, leading to infection and disease. The virulence factors that participate in the pathogenesis of respiratory infections include the lipopolysaccharide(LPS), the type III secretion system (T3SS), pyocyanin, and rhamnolipid (15). Among the numerous virulence factors, T3SS plays a key role in disrupting the host immune system during the acute infection of P. aeruginosa (16 –18).

T3SS is a needle-like complex of Gram-negative pathogens that includes five different components: needle structure, translocation apparatus, regulation system, effector protein, and chaperone protein (19). And the regulation of T3SS is a complex balance between different regulators (20). ExsA is the master regulator involved in the transcriptional activation of all genes of T3SS. Especially in low Ca²⁺ environments or on contact with host cells, ExsA is massively activated, activating transcription of T3SS-related genes (20, 21). Activation of T3SS rapidly disrupts host cell membranes and tight junctions between cells, undermining the innate immune response to infection (22 –24). Previous studies have also confirmed that the severity of acute P. aeruginosa infection and high mortality rates are strongly associated with the expression of T3SS (25, 26).

The quorum-sensing system, two-component system, and small regulatory RNA (sRNA) are known virulence regulatory systems (27). Recent studies have shown that sRNAs are the primary molecules that regulate the expression of many genes and virulence factors to help pathogens adapt rapidly to the host environment (28, 29). The sRNA PrrH in P. aeruginosa is only encoded by two adjacent tandem genes, PrrF1 and PrrF2. Previous studies showed that PrrH is functionally homologous to the RyhB RNAs encoded by Escherichia coli. PrrH and RyhB RNAs are mainly involved in iron and heme metabolism (30 –33). Subsequent studies showed that PrrH regulates the expression of virulence factors (31). And our previous study also showed that PrrH repressed the formation of pyocyanin, rhamnolipids, elastase, and biofilms (34). However, previous studies have shown that the prrH-deficient mutant attenuates the virulence in an acute lung infection model in mice. All mice infected with ΔprrH survived the entire 28-day course of the experiment, whereas all mice infected with the wild-type or the complemented mutant died of lung infection within 4 days of injection (31, 32), suggesting that PrrH may influence the pathogenicity through the regulation of other virulence factors. But the specific mechanism is not clear, Herein, we sought to explore the role of PrrH on bacterial-host interactions in acute lung infections.

PrrH enhances P. aeruginosa cytotoxicity and causes severe lung damage

An in vivo acute lung infection model in which C57BL/6 mice were infected with PAO1 for 6 h and 10 h was used to investigate changes in the expression of PrrH in P. aeruginosa during lung infection. Bacteria from alveolar lavage fluid were collected, and PrrH was examined using quantitative real time polymerase chain reaction (qRT-PCR). The expression levels of PrrH in the 6-h and 10-h groups were 3.84-fold and 8.76-fold higher, respectively, than in the uninfected group (Fig. 1A). These results suggested that the expression of PrrH in P. aeruginosa increased during lung infection.

Fig 1 — The effect of PrrH on *P. aeruginosa* pathogenicity, lung damage, and innate host immune response. (A) The C57BL/6 mice were infected with PAO1 strain at 1 × 10⁸ CFU, and the bacteria were extracted from alveolar lavage fluid 6 h and 10 h post-infection. PrrH expression was analyzed using qRT-PCR (Pre, uninfected group as control). (B) Survival curves of C57BL/6 mice infected with different *P. aeruginosa* strains (WT/Vector, Δ*prrH*/Vector, WT/PrrH, and Δ*prrH*/PrrH) at 3 × 10⁷ colony-forming units (CFU). (C) The C57BL/6 mice were infected with different *P. aeruginosa* strains at 3 × 10⁷ CFU for 24 h. The lung homogenate was centrifuged, and the supernatant was applied to the plate to culture and count the number of live CFU in the lung. (D) Lung anatomy of C57BL/6 mice infected with different *P. aeruginosa* strains at 3 × 10⁷ CFU for 24 h. (E) Hematoxylin and eosin staining of the lungs of C57BL/6 mice infected with different *P. aeruginosa* strains at 3 × 10⁷ CFU for 24 h. Observed under microscope, first row magnification 1.0×; second row magnification 10.0×. (F) Microscopic images of A549 cells infected with MOI50 of different *P. aeruginosa* strains for 12 h. (G) A549 cells were infected with different *P. aeruginosa* strains at MOI50 for 8 h. All bacteria in the co-culture system were recovered and counted on agar plates containing gentamicin (30 µg/mL). (H) A549 cells were infected with different strains at MOI50 for 12 h. The relative levels of lactate dehydrogenase in the co-culture supernatant were measured in response to the mortality of A549 cells. Values were expressed as the mean of at least three independent experiments ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant). MOI, multiplicity of infection.

As previously described (34), the wild-type strain (WT/Vector), the prrH gene-deficient strain (ΔprrH/Vector), the prrH-overexpression strain (WT/PrrH), and the prrH complementation strain, which overexpress the prrH gene in the background of ΔprrH mutation (ΔprrH/PrrH), were constructed. The expression levels of the PrrH in these strains were analyzed using qRT-PCR (Fig. S1). There was no significant difference in the growth of these strains (Fig. S2). To explore the function of PrrH in P. aeruginosa during lung infections, these strains were intranasally inoculated into C57BL/6 mice at 3 × 10⁷colony-forming units (CFU)/mouse. The weight and physiological state of the mice were recorded daily. The result was that none of the mice infected with the ΔprrH/Vector strain died after 8 days, 50% of mice infected with the WT/PrrH strain were alive after 3 days, and 25% of mice infected with the ΔprrH/PrrH strain were alive after 6 days (Fig. 1B). The WT/PrrH strain and ΔprrH/PrrH strain showed higher virulence in mouse lung infections. Then, mice were further dissected at 24 h post-infection, and the bacterial load in the lungs was measured. Compared to the WT/Vector, the ΔprrH/Vector strain had a lower bacterial load in the lung, whereas the WT/PrrH and ΔprrH/PrrH strains had a higher bacterial load (Fig. 1C). After infection with the WT/Vector, the lung developed edema and hemorrhage, which were mild in the lung of mice infected with the ΔprrH/Vector and more severe in the lung of mice infected with the WT/PrrH and ΔprrH/PrrH strain (Fig. 1D). The PrrH showed more pathogenicity, consistent with previous results. To determine the effect of PrrH on the host immune response, the mice were injected with different P. aeruginosa strains and the lung tissues were stained with hematoxylin and eosin (H&E), and the inflammatory response was observed. The WT/PrrH-infected mice and the ΔprrH/PrrH-infected mice showed a higher neutrophil infiltration and inflammatory response (Fig. 1E).

Next, the virulence of the different strains infected with A549 cells was then compared, and a similar phenomenon was observed. After infection for 12 h, most of the cells infected with the WT/Vector strain maintained their normal morphology, few cells were round and refractive, changing from an adnate to a suspended state. The cells infected with the ΔprrH/Vector strain maintained their normal morphology and even proliferated. However, the cells infected with the WT/PrrH and ΔprrH/PrrH strains were crinkled, rounded, refractive, and floated in the supernatant (Fig. 1F). To quantify the mortality of A549 cells, we assayed the relative release of lactate dehydrogenase in the co-culture supernatant. Compared with the WT/Vector strain, the bacterial load and cytotoxicity were lower for the ΔprrH/Vector after infection of A549 cells. The bacterial load and cytotoxicity were higher in the WT/PrrH and ΔprrH/PrrH strains (Fig. 1G and H).

Overall, in vivo mouse and in vitro cellular experiments confirmed that PrrH knockout attenuates P. aeruginosa pathogenicity and growth during infection and induces a mild immune response. In contrast, overexpression of PrrH enhances bacterial pathogenicity, accelerates growth during infection, and induces a stronger inflammatory response in the host.

PrrH promotes the expression of T3SS-related genes and targets ExsA

The primary pathogenic mechanism of P. aeruginosa is the secretion of various virulence factors that mediate escape from the host immune response. To investigate which virulence factors are controlled by PrrH, we examined the expression of multiple virulence factors in PrrH mutant strains. The quorum sensing (QS)-related virulence factors such as pyocyanin, elastase, rhamnolipid, and biofilm, which are negatively regulated by PrrH, were tested in a previous study (34). But the expression of these virulence factors was not compatible with PrrH-mediated cytotoxicity. Lipopolysaccharide (LPS) is a classical structural component of the outer membrane of most Gram-negative bacteria and is a potent agonist of the innate inflammatory response. We examined the expression of LPS in different PrrH mutant strains. However, LPS expression did not differ significantly among strains (Fig. S3), nor did the expression of LPS-related genes (Fig. S4).

The prediction analysis for the potential targets of PrrH based on the data in the IntaRNA database is shown in Table S1. Notably, we found that PrrH targets multiple T3SS-related genes, including the translocation apparatus, the basal body, the needle filament, the effector proteins, the regulation system, and the chaperones. We examined the expression of the representative T3SS-related genes (exsA, exoS, exoT, exoY, popB, popD, pcrV, and pscL) in different strains using qRT-PCR. The results showed that the expression of T3SS-related genes was significantly higher in the WT/PrrH and ΔprrH/PrrH strains compared to the WT/Vector strain (Fig. 2A). These results suggested that PrrH promotes the expression of T3SS-related genes. ExsA is a general transcriptional activator of all structural genes of the T3SS. In in vivo mouse experiments, the expression of both ExsA and PrrH was enhanced in P. aeruginosa pulmonary infection, which led us to explore the relationship between PrrH and ExsA. (Fig. 2B).

We then predicted the specific binding region of sRNA PrrH-mRNA ExsA using IntaRNA (Fig. 2C). The 24 nt sequence of PrrH predicted to bind to ExsA was located within the intergenic region of PrrH (PrrH_IG) (Table S4). First, we constructed different plasmids: the pSTV28-PrrH plasmid (expressing PrrH), the pSTV28-mut-PrrH plasmid (expressing PrrH with reverse mutation of the predicted binding site), pUCP32T-ExsA-green fluorescent protein (GFP) plasmid (expressing ExsA fragment containing the predicted binding site), and pUCP32T-mut-ExsA-GFP plasmid (expressing ExsA fragment containing the reverse mutation of the predicted binding site) (Fig. 2D). The predicted binding sequences and mutated sequences are shown in Fig. 2D and Table S4. Then, different plasmids were introduced into E. coli DH5α to construct different GFP reporter systems (Fig. 2E), the unmutated strains “PrrH/ExsA” (pSTV28-PrrH, pUCP32T-ExsA-GFP), the target-mutant strains “PrrH/Mut-ExsA” (pSTV28-PrrH, pUCP32T-mut-ExsA-GFP), and the sRNA-mutant strains “Mut-PrrH/ExsA” (pSTV28-mut-PrrH, pUCP32T-ExsA-GFP). We examined the GFP fluorescence intensity values in different strains, and the results showed that the fluorescence intensity of the PrrH/ExsA strain is stronger compared to the Mut-PrrH/ExsA and the PrrH/Mut-ExsA strains, both when viewed under a microscope and when detected by an enzyme marker. This phenomenon suggests that PrrH can bind to the predicted binding site of ExsA by base complementary pairing, affecting the enhancement of GFP fluorescence intensity.

PrrH increases the pathogenicity of P. aeruginosa by regulating ExsA

PrrH enhanced the pathogenicity of P. aeruginosa, causing severe lung damage, and promoted the expression of T3SS-related genes by targeting binding to ExsA. To explore whether PrrH regulates the cytotoxicity of P. aeruginosa primarily through the regulation of ExsA, an exsA gene-deficient strain in PAO1 (ΔexsA/Vector) and a prrH-overexpression strain in the ΔexsA mutation background (ΔexsA/PrrH) were constructed. The expression levels of T3SS-related genes in these strains were measured by qRT-PCR. The results showed that knockout of exsA in PAO1 significantly reduced the expression of T3SS-related genes. (Fig. 3A). And bacterial growth was not affected by knocking out the exsA gene. (Fig. 3B).

Fig 3 — The mechanism by which PrrH increases the pathogenicity of *P. aeruginosa*. (A) Validation of T3SS-related gene expression in mutant strains (WT/Vector, WT/PrrH, ΔexsA/Vector, and ΔexsA/PrrH) using qRT-PCR. (B) Knockout of the ExsA gene in PAO1 had no effect on the growth of *P. aeruginosa*. (C) The C57BL/6 mice were infected with different strains at 3 × 10⁷ CFU, and the daily mortality of the mice was observed and recorded (survival rate). (D) The C57BL/6 mice were infected with different strains at 3 × 10⁷ CFU for 24 h. The lung homogenate was centrifuged, and the supernatant was applied to the plate to culture and count the number of live CFU in the lung. (E) H&E staining of the lungs of C57BL/6 mice infected with different strains at 3 × 10⁷ CFU for 24 h. (F) Lung anatomy of C57BL/6 mice infected with different *P. aeruginosa* strains at 3 × 10⁷ CFU for 24 h. (G) A549 cells were infected with different *P. aeruginosa* strains at MOI50 for 12 h and the relative levels of lactate dehydrogenase in the co-culture supernatant were measured in response to the mortality of A549 cells. The values represent the mean of at least three independent experiments ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). MOI, multiplicity of infection.

To evaluate the cytotoxicity of the ΔexsA/Vector and ΔexsA/PrrH strains, corresponding strains were intranasally injected into C57BL/6 mice at 3 × 10⁷ CFU/mouse. The results showed that the group of mice infected with the WT/PrrH strain had a high mortality rate. However, none of the mice infected with the ΔexsA/Vector strain died, and only one mouse infected with the ΔexsA/PrrH died on the 5th day (Fig. 3C). And the bacterial loads in the lung were low for strains with ΔexsA/Vector and ΔexsA/PrrH strains (Fig. 3D). Then, to detect the host immune response induced by the ΔexsA/Vector and ΔexsA/PrrH strains, we performed H&E staining on the lung tissue. The lung tissue sections infected with the ΔexsA/Vector and ΔexsA/PrrH strains were pale, implying low neutrophil infiltration and weak inflammatory response (Fig. 3E). After dissecting the mice, we found lung tissue edema and hemorrhage in the lungs were slighter for mice infected with ΔexsA/Vector and ΔexsA/PrrH strains (Fig. 3F). These results suggested that knockout of exsA significantly reduced the pathogenicity of P. aeruginosa, and overexpression of prrH in the exsA mutant background did not restore the virulence of the strain. A similar phenomenon was observed in A549 cells, where the ΔexsA/Vector and ΔexsA/PrrH strains showed weak cytotoxicity (Fig. 3G).

Consequentlly, in vivo mouse and in vitro cellular experiments confirmed that knocking out exsA significantly reduced the cytotoxicity of P. aeruginosa, and the overexpression of PrrH in the ΔexsA did not also restore the virulence of the strain. This suggests that PrrH enhanced the cytotoxicity of P. aeruginosa primarily through the regulation of ExsA.

PrrH promotes host inflammatory response and apoptosis by regulating ExsA

PrrH exacerbated the lung injury and the inflammatory response of the host. Accordingly, we measured the expression levels of several inflammatory cytokines in mouse lung homogenates to assess the host inflammatory response caused by PrrH. Compared with WT/Vector strain, infection with the WT/PrrH and the ΔprrH/PrrH strains increased the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). However, infection with the ΔprrH/Vector, ΔexsA/Vector, and the ΔexsA/PrrH strains suppressed the expression of TNF-α and IL-1β (Fig. 4A). There was no significant difference in inflammatory factor interleukin-6 (IL-6) levels between the strains except for the ΔexsA/Vector and ΔexsA/PrrH strains. In vivo results revealed that PrrH induced inflammation in the host by primarily upregulating the secretion of TNF-α and IL-1β. Infection with the ΔexsA/Vector strains resulted in reduced expression of inflammatory factors, and infection with the ΔexsA/PrrH strains did not increase inflammatory factor expression, suggesting that PrrH-mediated inflammation is indeed mediated by modulation of ExsA. We also examined the expression of inflammatory factors in the A549 cells. Compared to the uninfected group, the cells infected with the WT/PrrH and the ΔprrH/PrrH strains increased the expression of TNF-α expressed by 50-fold, IL-6 by 30-fold, and IL-8 by 40-fold. The cells infected with the ΔprrH/Vector, ΔexsA/Vector, and ΔexsA/PrrH strains increased the expression of TNF, IL-6, and IL-8 by about 10-fold (Fig. 4B). Similarly, infection with the ΔexsA/PrrH strains did not increase the expression of inflammatory factors, a phenomenon consistent with that in mice. To investigate the mechanism of PrrH-mediated cell death, we examined the apoptosis rate of A549 cells infected with different P. aeruginosa strains. Compared with the WT group (9.87%), the apoptosis rate (6.04%) was lower in A549 cells infected with ΔprrH/Vector strain. However, the apoptosis rate was higher in A549 cells infected with WT/PrrH and ΔprrH/PrrH strains (18.84%; 18.15%). The apoptosis rates were lower for A549 cells infected with ΔexsA/Vector and ΔexsA/PrrH strains (11.29%; 7.78%) (Fig. 4C). Generally, PrrH enhanced the host inflammatory response and exacerbated apoptosis in A549 cells.

Fig 4 — PrrH promotes host inflammatory response and apoptosis . (A)The effect of different *P. aeruginosa* strains on inflammation. C57BL/6 mice were infected with different *P. aeruginosa* strains at 3 × 10⁷CFU. The expression of inflammatory factors (IL-1β, IL-6, and TNF-α) in the lungs was detected using the ELISA kit (compared with WT/Vector group). (B)A549 cells infected with MOI50 of different *P. aeruginosa* strains for 4 h. The expression of inflammatory factors (IL-8, IL-6, and TNF-α) was detected using qRT-PCR (compared with the uninfected group). (C)A549 cells infected with different *P. aeruginosa* strains for 8 h. The apoptosis was detected using flow cytometry. Values were expressed as the mean of at least three independent experiments ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant). MOI, multiplicity of infection.

Overall, these results show that PrrH induces severe lung damage, inflammation, and apoptosis by positively regulating ExsA expression.

DISCUSSION

P. aeruginosa is the leading Gram-negative cause of nosocomial pneumonia (1 –6). P. aeruginosa relies primarily on the secretion of large amounts of virulence factors to evade host immunity and successfully infect and cause disease (15, 35). In this study, we found that the expression of PrrH is increased during acute lung infection and that it causes severe lung injury and hemorrhage. Furthermore, we found that PrrH enhanced P. aeruginosa-induced lung injury mainly through the regulation of ExsA, a master regulator of T3SS. In addition, PrrH enhanced the host inflammatory response and the apoptosis of epithelial cell. Thus, this study illustrated how PrrH helps to regulate bacterial pathogenicity and facilitates escape in P. aeruginosa lung infections.

Numerous previous studies have implicated sRNAs in the regulation of P. aeruginosa pathogenicity (21, 36 –38). PrrH was originally described as a “heme-responsive RNA” (31). Our previous study showed that PrrH repressed the formation of QS system-related virulence including pyocyanin, rhamnolipids, elastase, and biofilms (34). However, in an acute mice lung infection model, previous studies have shown that the prrH-deficient mutant attenuates virulence, but the specific mechanism is not clear (31, 32). In this study, we found that the strains overexpressing PrrH were highly cytotoxic and caused a significant injury and hemorrhage in lung. Therefore, we speculated that PrrH may influence pathogenicity by regulating other virulence factors. P. aeruginosa can adapt to the adverse host and contribute to successful infection by secreting a variety of virulence factors, including LPS, T3SS, T6SS, and biofilms (15, 35). The LPS is the endotoxin and inflammatory activator in Gram-negative bacteria (39). We examined LPS expression in several PrrH mutant strains and found that PrrH has no effect on both LPS protein synthesis and LPS-related gene expression (Fig. S3 and S4). We then analyzed the possible target genes of PrrH by IntaRNA prediction and found that it targets several genes related to the T3SS. Therefore, we further validated the relationship of PrrH and T3SS.

T3SS is a complex bacterial machinery consisting of five distinct components: needle structure; translocation apparatus; regulatory system; chaperones and effectors (19). In previous studies, the expression of T3SS had been proven that it can be directly regulated by multiple regulators (e.g., expressor PsrA and repressor PtrA/AtrR) and other relative material which participate in regulating the T3SS (e.g., PsrA/RpoS cAMP/Vfr, GacSA-RsmYZ-RsmA system, and VqsM, RetS, LadS, MvaT, and MvaU) (19). The IntaRNA prediction results showed that PrrH targets several structural T3SS-related genes (Table S1). We further selected representative genes from the five components and examined the expression of these genes in mutant strains of PrrH (Fig. 2A). The results showed that the expression of all T3SS-related genes was significantly higher in the prrH-overexpressing strains. One of them, ExsA, is a universal T3SS regulator. It is involved in transcriptionally activating the five operons encoding all structural and the five regulatory genes (40, 41). Therefore, our hypothesis is that PrrH may activate the T3SS through regulation of the transcription activator ExsA. The GFP fluorescent reporter assays proved that PrrH does indeed bind to ExsA mRNA. Based on the IntaRNA prediction, it is possible that PrrH also targets other T3SS-related genes. The specific regulatory relationships of PrrH and related genes can be subsequently verified by qRT-PCR and the GFP fluorescent reporter systems.

The results show that compared to the PrrH/Mut-ExsA strains and the Mut-PrrH/ExsA strains, the fluorescence intensity of GFP significantly enhanced in PrrH/ExsA strain (Fig. 2F). As our guess, when PrrH binds to the predicted sequence of ExsA, it may open the hairpin structure and promotes translation of GFP, resulting in bright fluorescence. The results also show that the PrrH_IG sequence is essential for binding and potentially activating ExsA translation.

T3SS is an important virulence factor in the acute phase of P. aeruginosa infection. It can activate multiple host immune pathways. The effector ExoT activates the mitochondrial/cytochrome c-dependent apoptotic pathway and effectively blocks necrotic cell death in Hela cells (17). The formation of a translocation competent T3SS is essential for the triggering of caspase-1 activation and IL-1β maturation (18). The effectors ExoS and ExoT regulated the expression of genes associated with acute and chronic infections in an nuclear factor-kappa B (NF-κB)-dependent manner (42). In our study, we found that PrrH significantly enhanced the host inflammatory response (Fig. 2E), and we demonstrated that the upregulation of inflammatory factor expression by PrrH and the increased rate of apoptosis were achieved mainly by regulating ExsA (Fig. 4A and B). These findings are consistent with the previously reported T3SS-mediated immune response of the host.

An in vivo acute lung infection model demonstrated that the expression of PrrH in P. aeruginosa increased during lung infection (Fig. 1A). It has previously been reported that prrH is an iron ion and heme-responsive sRNA (30, 31). The ferric uptake regulator can bind to the promoter region of prrH to regulate prrH expression (43). Therefore, we hypothesized that during acute lung infection, hemorrhage from lung injury releases large amounts of heme from iron ions, which affects prrH expression. Thus, PrrH is a sensor that rapidly responds to environmental changes, activating downstream virulence factors to enhance virulence and help P. aeruginosa escape.

In summary, our study revealed that the expression of PrrH is elevated during P. aeruginosa lung infection, which mediates severe lung injury by regulating the T3SS activator ExsA. This function of PrrH may provide a potential target for the clinical treatment of P. aeruginosa infections, especially T3SS-positive bacteria.

MATERIALS AND METHODS

Bacterial strains and culture conditions

The PAO1 strain was donated by Professor Zhou (Children’s Hospital of Chongqing Medical University). The strains were cultured in fresh Luria-Bertani (LB) medium or LB plates (1.5% agar) at 37°C. If needed, antibiotics were added to the medium or plates at the following concentrations: 30 µg/mL gentamicin and 16 µg/mL chloramphenicol. The construction of PAO1 prrH-deficient mutants was performed in our previous study. Details of the strains are provided in Table S2.

Construction of the exsA-deficient mutants in PAO1

The sacB-based suicide vector system was used for the knockout of exsA in PAO1, as described in our previous study (34). Briefly, the upstream and downstream sequences of the exsA, termed flanking fragment A and flanking fragment B, were amplified by fusion PCR and ligated to form a recombinant fragment AB. The AB fragment was cloned into XbaI/SacI sites in pGSM to generate the recombinant plasmid pGSM-ΔexsA and was then transformed into PAO1 to generate exsA mutant strain (ΔexsA). An empty plasmid (pROp200) and an overexpression plasmid (pROp200-prrH) were transformed into ΔexsA to generate exsA mutant strains with empty plasmids (ΔexsA/Vector) and exsA mutant strains with a prrH-overexpression plasmid (ΔexsA/PrrH), respectively.

Cell culture and infection

Human pulmonary epithelial cell line A549 (ATCC: CCL-185) cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) (Gibco, Carlsbad, CA) containing 10% Fetal Bovine Serum (FBS) (Gibco, Carlsbad, CA) at 37°C under 5% CO₂. Cells were passaged at a ratio of 1:5 each time and cultured continuously for 3 days. A549 cells were seeded in well plates and incubated at 37°C for 14 h. The indicated strains underwent a logarithmic growth phase and were collected from the solution and resuspended in phosphate buffered solution (PBS). Prior to infection, the original cell culture medium was removed and replaced with DMEM containing 1% FBS. Cells were infected with bacteria at a specific dose based on the absorbance of the suspensions at 600 nm (OD₆₀₀). Bacterial CFU and OD₆₀₀ values were obtained using the formula 22.031 × OD₆₀₀ + 0.8278 = CFU (unit: 10⁸ CFU/mL).

Mouse infections

C57BL/6 mice (male, 6 weeks old, weighing 17 g–19 g) were purchased from Guangdong Animal Centre. The indicated strains, which underwent logarithmic growth phases were removed from the solution and resuspended in PBS. Appropriate dilutions were prepared according to the OD₆₀₀. Mice were anesthetized with isoflurane (4%) and intranasally inoculated with 30µL of bacterial solution. Mice were infected at a dose of 1 × 10⁸CFU and divided into two infection groups (seven mice per group) according to different infection times (6h and 10h). The PAO1 bacteria in the alveolar lavage fluid of the mice were collected and tested simultaneously with uninfected strains for PrrH gene expression. The infectious dose used in the survival curve was 3 × 10⁷CFU (lethal dose 50%, LD50) per mouse (eight mice for each bacterial strain). Body weight and mortality of the mice were observed and recorded daily. Survival curves were plotted based on mortality in each group within 7 days of infection. Mice were infected with a dose of 3 × 10⁷CFU and euthanized after 24 h. Lung tissues and lungs were homogenized for gene expression and CFU count assays. H&E staining of mouse lung tissues was performed to observe inflammatory infiltration in the lungs. Briefly, lung tissues were fixed in 4% paraformaldehyde (Biosharp, China) overnight, dehydrated in ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.

Colony-Forming Units

For in vivo mice experiments, lung tissues were ground into lung homogenates, and supernatants were collected after centrifugation at 1,000 rpm and then diluted with PBS. Then, 10 µL of the diluted solution was titrated onto a gentamicin-resistant plate (30 µg/mL) and incubated at 37°C for 16 h. The number of monoclonal clones on the plate was counted and converted into the original bacterial load of the lung homogenate with a dilution multiple. For in vitro cellular experiments, A549 cells were seeded on a six-well plate (8 × 10⁵/well) for 1 day and then infected with the indicated strains at a multiplicity of infection (MOI) of 50 for 8 h. The supernatant from the co-culture system was removed and 2 mL of 0.1% Triton X-100 was added to the well plate to completely lyse the cells and release the bacteria. The bacteria in the supernatant and the released bacteria were collected and diluted with PBS. Fifty microliters of the diluted bacterial solution was applied to a gentamicin-resistant plate (30 µg/mL) and incubated at 37°C for 16 h. The number of monoclonal clones was counted and converted to the bacterial load in a co-culture system based on a dilution multiple.

LPS assay

A centrifuge can be used at 13,000 rpm at room temperature to harvest 2 mL–5 mL of bacterial cells. LPS is extracted from concentrated bacteria by kit (iNtRON Biotechnology), and added to 30 μL–50 μL of 10mM Tris-HCl buffer to fully dissolve it. Then the LPS concentration was measured by the LPS ELISA kit.

Cytotoxicity assay

In principle, cytotoxicity assays are based on the quantitative analysis of cytotoxicity by measuring the activity of lactate dehydrogenase (LDH) released into the culture medium. Briefly, A549 cells were seeded on a 96-well plate (5 × 10⁴/well) 1 day before infection and infected with the indicated bacterial strains at an MOI of 50 at 37°C with 5% CO₂ for 12 h. Three control groups were established: cell-free culture wells (DMEM control), uninfected cell wells (cell control), and uninfected cell wells for maximum lysis (positive control). The LDH release reagent was added to the maximum lysis well for 1 h and the sample was collected according to the manufacturers’ instructions for the LDH cytotoxicity assay kit (Beyotime, Shanghai, China). The absorbance values at 490 nm (OD₄₉₀) and 900 nm (OD₉₀₀) were recorded simultaneously, and OD₉₀₀ was subtracted from OD₄₉₀. The percentage of cytotoxicity was calculated using the following formula: (A_co-culture − A_{cell control}) / (A_{positive control} − A_{cell control}) × 100%.

GFP reporter assays

The GFP reporter plasmid system (pUCP32T-gfp) was used for exsA according to a previously described method. The putative binding sites of PrrH targeting ExsA were predicted by IntaRNA. A wild-type fragment of ExsA mRNA containing putative binding sites for PrrH was amplified through PCR and inserted into the XbaI/NcoI sites upstream of the first codon of GFP in the pUCP32T-gfp plasmid to generate a translational fusion plasmid, pUCP32T-exsA-gfp. Similarly, a mutated mut-exsA fragment was inserted into the XbaI/NcoI site upstream of the first codon of GFP in the pUCP32T-gfp plasmid to generate a mutant plasmid pUCP32T-mut exsA-gfp. The pUCP32T-exsA-gfp or pUCP32T-mut exsA-gfp was then transformed into competent E. coli DH5α cells with pSTV28-prrH or pSTV28-mut PrrH. Three DH5α strains (PrrH/ExsA, PrrH/Mut-ExsA, Mut-PrrH/ExsA) were collected through centrifugation and resuspended in PBS. Absorbance (Abs600) and fluorescence intensity (F485/535) were measured using a BioTek Synergy H1 microplate reader (BioTek, Winooski, VT). The GFP activity was expressed in arbitrary units as F485/535/Abs600. In addition, 10 µL of the bacterial solution was pipetted onto the slide, and the fluorescence was observed using a Nikon ECLIPSE Ti2-U fluorescence microscope (Nikon, Tokyo, Japan).

Real-time PCR

Bacteria and cells were collected through centrifugation and lysed with the TRIzol Reagent (Takara Bio Inc.). Total RNA was extracted according to the kit instructions and quantified using a Nanotrap 2000 spectrophotometer. The RNA (1 µg) was reverse transcribed using the PrimeScript RT reagent kit (TaKaRa, Dalian, China). The cDNA was subjected to qPCR on a ViiATM 7 Dx system (Applied Biosystems, Foster, CA, USA) using SYBR green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China). The expression levels of the target genes were normalized to the expression of the reference genes (rpoD or β-actin) and analyzed using the relative threshold cycling (2^−ΔΔCt) method. The primer sequences used are listed in Table S3.

Cytokine assay

The production of cytokines in lung homogenates or cell supernatants was measured using an ELISA kit (MULTI SCIENCES, for IL-6 and TNF-α; Sangon Biotech, for IL-1β) according to the manufacturer’s instructions.

Flow cytometry for apoptosis

A549 cells inoculated into 12-well plates (4 × 10⁵/well) were infected with the indicated strain at an MOI of 50 for 8 h. Cells in the well plates were then digested with trypsin (0.25%) and collected by centrifugation. The collected cells were resuspended with 500 µL binding buffer according to the instructions of the Apoptosis Detection Kit (KeyGEN BioTECH, Jiangsu, China) and stained with 5 µL membrane-bound protein (Annexin V-FITC) and 5 µL propidium iodide for 15 min at room temperature. The stained cells were analyzed by flow cytometry (Novo Quanteon) within 1 h .

Statistical analysis

At least three independent experiments were performed for each separate set of assays. Data are presented as mean ± SEM using GraphPad Prism 9 (GraphPad Software, San Diego, CA). Statistical significance was determined by t-test between two groups, with P-values represented as *P, 0.05, **P, 0.01, and ***P, 0.001.

ACKNOWLEDGMENTS

This work was supported by the National Science Foundation of China (Grant No. 82072345; No. 82172329; No. 82202590), the Guangzhou Science Technology and Innovation Commission (Grant No. 202201011470), and Guangdong Provincial Hospital of Traditional Chinese Medicine (Grant No. YN2019MJ01).

We are very grateful to Prof. Shiyun Chen (Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China) for providing the plasmid pROp200.

Conceived and designed the experiments: C.C., B.H., J.P., and Q.S. Performed the experiments: Q.S. and S.Z. Wrote the paper: Q.S. Analyzed the data: R.Y., J.Z., and C.S. Contributed other analysis or discussion: M.L., X.Z., and C.Z.

Contributor Information

Bin Huang, Email: huangb3@mail.sysu.edu.cn.

Jieying Pu, Email: pujy1105@163.com.

Cha Chen, Email: chencha906@163.com.

Fernando Navarro-Garcia, Cinvestav-IPN, Mexico.

ETHICS APPROVAL

All animal experiments were approved for animal ethics by the Guangzhou University of Chinese Medicine.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.00626-23.

Supplemental material. spectrum.00626-23-s0001.pdf.

Fig. S1 to S4; Tables S1 to S4.

spectrum.00626-23-s0001.pdf^{(261.5KB, pdf)}

DOI: 10.1128/spectrum.00626-23.SuF1

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental material. spectrum.00626-23-s0001.pdf.

Fig. S1 to S4; Tables S1 to S4.

spectrum.00626-23-s0001.pdf^{(261.5KB, pdf)}

DOI: 10.1128/spectrum.00626-23.SuF1

[B1] 1. Luyt CE, Hékimian G, Koulenti D, Chastre J. 2018. Microbial cause of ICU-acquired pneumonia: hospital-acquired pneumonia versus ventilator-associated pneumonia. Curr Opin Crit Care 24:332–338. doi: 10.1097/MCC.0000000000000526 [DOI] [PubMed] [Google Scholar]

[B2] 2. Jang JH, Yeo HJ, Kim T, Cho WH, Min KH, Hong S-B, Baek A-R, Lee H-K, Kim C, Chang Y, Park HK, Oh JY, Lee HB, Bae S, Moon JY, Yoo KH, Gil H-I, Jeon K. 2022. Microbiologic pattern and clinical outcome of non-ICU-acquired pneumonia: Korean HAP registry analysis. Korean J Intern Med 37:800–810. doi: 10.3904/kjim.2021.348 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Naidus EL, Lasalvia MT, Marcantonio ER, Herzig SJ. 2018. The diagnostic yield of noninvasive microbiologic sputum sampling in a cohort of patients with clinically diagnosed hospital-acquired pneumonia. J Hosp Med 13:34–37. doi: 10.12788/jhm.2868 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Sano M, Shindo Y, Takahashi K, Okumura J, Sakakibara T, Murakami Y, Iguchi M, Yagi T, Matsui S, Hasegawa Y. 2022. Risk factors for antibiotic resistance in hospital-acquired and ventilator-associated pneumonia. J Infect Chemother 28:745–752. doi: 10.1016/j.jiac.2022.02.012 [DOI] [PubMed] [Google Scholar]

[B5] 5. Yin Y, Zhao C, Li H, Jin L, Wang Q, Wang R, Zhang Y, Zhang J, Wang H. 2021. Clinical and microbiological characteristics of adults with hospital-acquired pneumonia: a 10-year prospective observational study in China. Eur J Clin Microbiol Infect Dis 40:683–690. doi: 10.1007/s10096-020-04046-9 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Feng D-Y, Zhou Y-Q, Zou X-L, Zhou M, Wu W-B, Chen X-X, Wang Y-H, Zhang T-T. 2019. Factors influencing mortality in hospital-acquired pneumonia caused by gram-negative bacteria in China. J Infect Public Health 12:630–633. doi: 10.1016/j.jiph.2019.02.014 [DOI] [PubMed] [Google Scholar]

[B7] 7. Micek ST, Wunderink RG, Kollef MH, Chen C, Rello J, Chastre J, Antonelli M, Welte T, Clair B, Ostermann H, Calbo E, Torres A, Menichetti F, Schramm GE, Menon V. 2015. An international multicenter retrospective study of Pseudomonas aeruginosa nosocomial pneumonia: impact of multidrug resistance. Crit Care 19:219. doi: 10.1186/s13054-015-0926-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Borgatta B, Gattarello S, Mazo CA, Imbiscuso AT, Larrosa MN, Lujàn M, Rello J. 2017. The clinical significance of pneumonia in patients with respiratory specimens harbouring multidrug-resistant Pseudomonas aeruginosa: a 5-year retrospective study following 5667 patients in four general ICUs. Eur J Clin Microbiol Infect Dis 36:2155–2163. doi: 10.1007/s10096-017-3039-z [DOI] [PubMed] [Google Scholar]

[B9] 9. Poovieng J, Sakboonyarat B, Nasomsong W. 2022. Bacterial etiology and mortality rate in community-acquired pneumonia, healthcare-associated pneumonia and hospital-acquired pneumonia in Thai university hospital. Sci Rep 12:9004. doi: 10.1038/s41598-022-12904-z [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. Djordjevic ZM, Folic MM, Jankovic SM. 2017. Distribution and antibiotic susceptibility of pathogens isolated from adults with hospital-acquired and ventilator-associated pneumonia in intensive care unit. J Infect Public Health 10:740–744. doi: 10.1016/j.jiph.2016.11.016 [DOI] [PubMed] [Google Scholar]

[B11] 11. Meschiari M, Orlando G, Kaleci S, Bianco V, Sarti M, Venturelli C, Mussini C. 2021. Combined resistance to ceftolozane-tazobactam and ceftazidime-avibactam in extensively drug-resistant (XDR) and multidrug-resistant (MDR) Pseudomonas aeruginosa: resistance predictors and impact on clinical outcomes besides implications for antimicrobial stewardship programs. Antibiotics (Basel) 10:1224. doi: 10.3390/antibiotics10101224 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

The small RNA PrrH aggravates Pseudomonas aeruginosa-induced acute lung injury by regulating the type III secretion system activator ExsA

Qixuan Shi

Shenghe Zeng

Ruiqi Yu

Mo Li

Cong Shen

Xuan Zhang

Chanjing Zhao

Jianming Zeng

Bin Huang

Jieying Pu

Cha Chen

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

PrrH enhances P. aeruginosa cytotoxicity and causes severe lung damage

Fig 1.

PrrH promotes the expression of T3SS-related genes and targets ExsA

Fig 2.

PrrH increases the pathogenicity of P. aeruginosa by regulating ExsA

Fig 3.

PrrH promotes host inflammatory response and apoptosis by regulating ExsA

Fig 4.

DISCUSSION

MATERIALS AND METHODS

Bacterial strains and culture conditions

Construction of the exsA-deficient mutants in PAO1

Cell culture and infection

Mouse infections

Colony-Forming Units

LPS assay

Cytotoxicity assay

GFP reporter assays

Real-time PCR

Cytokine assay

Flow cytometry for apoptosis

Statistical analysis

ACKNOWLEDGMENTS

Contributor Information

ETHICS APPROVAL

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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