Polyamine-binding protein PotD2 is required for stress tolerance and virulence in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the cause of porcine contagious pleuropneumonia, which is one of the most important respiratory diseases in swine and causes huge economic losses in the swine industry. PotD, a polyamine-binding protein, has been well characterised in many pathogens of humans and animals. In this study, a ΔpotD2 mutant of A. pleuropneumoniae strain MS71 (serovar 1) was constructed successfully by homologous recombination. Growth curves of different strains showed that the growth of the ΔpotD2 mutant was affected greatly in the logarithmic phase compared with that of parental strain. In vitro stress assays revealed that the viability of ΔpotD2 mutant strain was significantly impaired under multiple environmental stresses, including high temperature, oxidation and hyperosmosis. Additionally, the ΔpotD2 mutant caused significantly decreased mortality in a mouse model. Taken together, the findings in this study suggest an important role of PotD2 in the growth, stress tolerance and virulence of A. pleuropneumoniae.

Deletion of potD, encoding a putative spermidine-binding protein, results in a complex phenotype in Legionella pneumophila.

L. pneumophila is an intracellular pathogen that replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV). We previously observed that the polyamine spermidine, produced by host cells or added exogenously, enhances the intracellular growth of L. pneumophila. To study this enhancing effect and determine whether polyamines are used as nutrients, we deleted potD from L. pneumophila strain JR32. The gene potD encodes a spermidine-binding protein that in other bacteria is essential for the function of the PotABCD polyamine transporter. Deletion of potD did not affect L. pneumophila growth in vitro in the presence or absence of spermidine and putrescine, suggesting that PotD plays a redundant or no role in polyamine uptake. However, deletion of potD resulted in a puzzlingly complex phenotype that included defects in L. pneumophila's ability to form filaments, tolerate Na(+), associate with macrophages and amoeba, recruit host vesicles to the LCV, and initiate intracellular growth. Moreover, the ΔpotD mutant was completely unable to grow in L929 cells treated with a pharmacological inhibitor of spermidine synthesis. These complex and disparate effects suggest that the L. pneumophila potD encodes either: (i) a multifunctional protein, (ii) a protein that interacts with, or regulates a, multifunctional protein, or (iii) a protein that contributes (directly or indirectly) to a regulatory network. Protein function studies with the L. pneumophila PotD protein are thus warranted.

Deletion of Polyamine Transport Protein PotD Exacerbates Virulence in Glaesserella (Haemophilus) parasuis in the Form of Non-biofilm-generated Bacteria in a Murine Acute Infection Model.

Polyamines are small, polycationic molecules with a hydrocarbon backbone and multiple amino groups required for optimal cell growth. The potD gene, belonging to the ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system, playing a pivotal role in bacterial metabolism and growth. The swine pathogen Glaesserella parasuis possesses an intact pot operon, and the studies presented here mainly examined the involvement of PotD in Glaesserella pathogenesis. A potD-deficient mutant was constructed using a virulent G. parasuis strain SC1401 by natural transformation; immuno-electron microscopy was used to identify the subcellular location of native PotD protein; an electron microscope was adopted to inspect biofilm and bacterial morphology; immunofluorescence technique was employed to study cellular adhesion, the levels of inflammation and apoptosis. The TSA++-pre-cultured mutant strain showed a significantly reduced adhesion capacity to PK-15 and MLE-12 cells. Likewise, we also found attenuation in virulence using murine models focusing on the clinical sign, H&E, and IFA for inflammation and apoptosis. However, when the mutant was grown in TSB++, virulence recovered to normal levels, along with a high level of radical oxygen species formation in the host. The expression of PotD could actively stimulate the production of ROS in Raw 264.7. Our data suggested that PotD from G. parasuis has a high binding potential to polyamine, and is essential for the full bacterial virulence within mouse models. However, the virulence of the potD mutant is highly dependent on its TSA++ culture conditions rather than on biofilm-formation.

The expansive effects of polyamines on the metabolism and virulence of Streptococcus pneumoniae.

Polyamines are common intracellular metabolites of nearly all cells, and their conservation across a vast diversity of cells suggests critical roles for these compounds in cellular physiology. Most intracellular polyamines are associated with RNA and, subsequently, polyamines have significant effects on transcription and translation. Putrescine and spermidine are the most common polyamines in bacteria. Intracellular polyamine pools in bacteria are tightly controlled by both de novo synthesis and transport. Polyamine homeostasis is emerging as a critical parameter of multiple pathways and physiology with substantial impact on bacterial pathogenesis, including the important human pathogen Streptococcus pneumoniae. Modulation of polyamine metabolism in pneumococci is an important regulator of central metabolism. It has broad effects on virulence factors such as capsule as well as stress responses that ultimately impact the survival of pneumococcus in a host. Polyamine transport protein as a single antigen or in combination with other pneumococcal proteins is shown to be an efficacious immunogen that protects against nasopharyngeal colonization, and invasive disease. A comprehensive description of polyamine metabolic pathways and their intersection with pneumococcal pathogenesis will undoubtedly point to novel approaches for treatment and prevention of pneumococcal disease.

Polyamine Transport Protein PotD Protects Mice against Haemophilus parasuis and Elevates the Secretion of Pro-Inflammatory Cytokines of Macrophage via JNK-MAPK and NF-κB Signal Pathways through TLR4.

The potD gene, belonging to the well-conserved ABC (ATP-binding cassette) transport system potABCD, encodes the bacterial substrate-binding subunit of the polyamine transport system. In this study, we found PotD in Haemophilus (Glaesserella) parasuis could actively stimulate both humoral immune and cellular immune responses and elevate lymphocyte proliferation, thus eliciting a Th1-type immune response in a murine immunity and infection model. Stimulation of Raw 264.7 macrophages with PotD validated that Toll-like receptor 4, rather than 2, participated in the positive transcription and expression of pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α using qPCR and ELISA. Blocking signal-regulated JNK-MAPK and RelA(p65) pathways significantly decreased PotD-induced pro-inflammatory cytokine production. Overall, we conclude that vaccination of PotD could induce both humoral and cellular immune responses and provide immunoprotection against H. parasuis challenge. The data also suggest that Glaesserella PotD is a novel pro-inflammatory mediator and induces TLR4-dependent pro-inflammatory activity in Raw 264.7 macrophages through JNK-MAPK and RelA(p65) pathways.

Systemic immunization with rPotD reduces Streptococcus pneumoniae nasopharyngeal colonization in mice.

Streptococcus pneumoniae (pneumococcus) is a human pathogen that can cause otitis media, pneumonia and, in severe cases, meningitis and bacteremia. The pneumococcus expresses PotD, a protein belonging to the polyamines transporter complex called PotABCD. PotD is a membrane-associated protein that binds polyamines and has been shown to be important for virulence. In this work we demonstrate that subcutaneous immunization with rPotD reduces the bacterial load in the nasal tissue of mice, following intranasal challenge with a type 6B pneumococcus. The protective effect correlated with the induction of high levels of antibodies in the immunized group; the antibodies were able to increase bacterial phagocytosis by mouse peritoneal cells. The cellular immune response was characterized by the production of gamma-interferon, IL-2 and IL-17 by splenocytes and nitric oxide by peritoneal cells of immunized mice, upon stimulation with rPotD. Taken together our results suggest that PotD is a promising candidate to be included in a protein based pneumococcal vaccine, able to induce phagocytic antibodies, a Th1 cellular immune response and production of IL-17, reducing nasopharyngeal colonization, the main event responsible for transmission of pneumococci in humans.