TroR Negatively Regulates the TroABCD System and Is Required for Resistance to Metal Toxicity and Virulence in Streptococcus suis.

Streptococcus suis is an emerging zoonotic pathogen that causes severe swine and human infections. Metals are essential nutrients for life; however, excess metals are toxic to bacteria. Therefore, maintenance of intracellular metal homeostasis is important for bacterial survival. Here, we characterize a DtxR family metalloregulator, TroR, in S. suis. TroR is located upstream of the troABCD operon, whose expression was found to be significantly downregulated in response to excess manganese (Mn). Deletion of troR resulted in reduced growth when S. suis was cultured in metal-replete medium supplemented with elevated concentrations of zinc (Zn), copper (Cu), or cobalt (Co). Mn supplementation could alleviate the growth defects of the ΔtroR mutant under Zn and Co excess conditions; however, it impaired the growth of the wild-type (WT) and complemented (CΔtroR) strains under Cu excess conditions. The growth of ΔtroR was also inhibited in metal-depleted medium supplemented with elevated concentrations of Mn. Moreover, the ΔtroR mutant accumulated increased levels of intracellular Mn and Co, rather than Zn and Cu. Deletion of troR in S. suis led to significant upregulation of the troABCD operon. Furthermore, troA expression in the WT strain was induced by ferrous iron [Fe(II)] and Co and repressed by Mn and Cu; the repression of troA was mediated by TroR. Finally, TroR is required for S. suis virulence in an intranasal mouse model. Together, these data suggest that TroR is a negative regulator of the TroABCD system and contributes to resistance to metal toxicity and virulence in S. suis. IMPORTANCE Metals are essential nutrients for life; however, the accumulation of excess metals in cells can be toxic to bacteria. In the present study, we identified a metalloregulator, TroR, in Streptococcus suis, which is an emerging zoonotic pathogen. In contrast to the observations in other species that TroR homologs usually contribute to the maintenance of homeostasis of one or two metals, we demonstrated that TroR is required for resistance to the toxicity conferred by multiple metals in S. suis. We also found that deletion of troR resulted in significant upregulation of the troABCD operon, which has been demonstrated to be involved in manganese acquisition in S. suis. Moreover, we demonstrated that TroR is required for the virulence of S. suis in an intranasal mouse model. Collectively, these results suggest that TroR is a negative regulator of the TroABCD system and contributes to resistance to metal toxicity and virulence in S. suis.

Amentoflavone Ameliorates Streptococcus suis-Induced Infection In Vitro and In Vivo.

Streptococcus suis, an important zoonotic pathogen, has caused considerable economic losses in the swine industry and severe public health issues worldwide. The development of a novel effective strategy for the prevention and therapy of S. suis is urgently needed. Here, amentoflavone, a natural biflavonoid compound isolated from Chinese herbs that has negligible anti-S. suis activity, was identified as a potent antagonist of suilysin (SLY)-mediated hemolysis without interfering with the expression of SLY. Amentoflavone effectively inhibited SLY oligomerization, which is critical for its pore-forming activity. The treatment with amentoflavone reduced S. suis-induced cytotoxicity in macrophages (J774 cells). Furthermore, S. suis-infected mice that received amentoflavone exhibited lower mortality and bacterial burden. Additionally, amentoflavone significantly decreased the production of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6 in an S. suis-infected cell model. Analyses of signaling pathways demonstrated that amentoflavone reduced S. suis-induced inflammation in S. suis serotype 2 (SS2)-infected cells by regulating the p38, Jun N-terminal protein kinase 1 and 2 (JNK1/2), and NF-κB pathways. The antivirulence and anti-inflammatory properties of amentoflavone against S. suis infection provide the possibility for future pharmaceutical application of amentoflavone in the treatment of S. suis infection.IMPORTANCE The widespread use of antibiotics in therapy and in the prevention of Streptococcus suis infection in the swine industry raises concerns for the emergence of a resistant strain. The use of antivirulence agents has potential benefits, mainly because of the reduced selective pressure for the development of bacterial resistance. In this study, we found that amentoflavone is an effective agent against S. suis serotype 2 (SS2) infection both in vitro and in vivo Our results demonstrated that amentoflavone is a promising anti-infective therapeutic for S. suis infections, due to its antivirulence and anti-inflammatory effects without antibacterial activity, with fewer side effects than conventional antibacterial agents.

Comparative immunosecretome analysis of prevalent Streptococcus suis serotypes.

Streptococcus suis is a major Gram-positive swine pathogen associated with a wide variety of diseases in pigs. The efforts made to develop vaccines against this pathogen have failed because of lack of common cross-reactive antigens against different serotypes. Nowadays the interest has moved to surface and secreted proteins, as they have the highest chances to raise an effective immune response because they are in direct contact with host cells and are really exposed and accessible to antibodies. In this work, we have performed a comparative immunosecretomic approach to identify a set of immunoreactive secreted proteins common to the most prevalent serotypes of S. suis. Among the 67 proteins identified, three (SSU0020, SSU0934, and SSU0215) were those predicted extracellular proteins most widely found within the studied serotypes. These immunoreactive proteins may be interesting targets for future vaccine development as they could provide possible cross-reactivity among different serotypes of this pathogen.

Antimicrobial activity of selected essential oils against Streptococcus suis isolated from pigs.

The inhibitory potential by contact and vapor of basil, cinnamon, clove, peppermint, oregano, rosemary, common thyme, and red thyme essential oils (EOs) against 20 strains of Streptococcus suis was determined by the disk diffusion test. The broth microdilution method was used to determine the minimal inhibitory and minimal bactericidal concentration (MIC and MBC) of the four selected oils. Furthermore, the bactericidal power (ratio MBC/MIC) was calculated. The EOs with the major potential in the disk diffusion method were red thyme, common thyme, oregano, and cinnamon (∅ mean 16.5-34.2 mm), whereas cinnamon did not show vapor activity. In the microdilution test, all the EOs showed notable antimicrobial activity (MIC90 and MBC90 312.5-625 µg·ml-1 ) and a strong bactericidal power (ratio = 1). This is the first study that selects essential oils against S. suis. New studies about the possible synergic effect of EOs with antibiotics and about toxicity and efficacy in in vivo conditions are recommended.

The manganese efflux system MntE contributes to the virulence of Streptococcus suis serotype 2.

Manganese is an essential micronutrient to bacteria and plays an important role in bacterial physiology. However, an excess of manganese is extremely deleterious to the cell. The manganese efflux system is used to control intracellular manganese levels by some bacteria. In this study, we have identified a cation efflux family protein (MntE) that functions as a manganese export system in Streptococcus suis serotype 2. To investigate the role of mntE in S. suis 2, a mntE deletion mutant (ΔmntE) and the corresponding complementation strain (CΔmntE) were constructed. ΔmntE displayed similar growth compared to the wild-type and complementation strains under normal growth conditions, but was defective in medium supplemented with high concentrations of manganese. In addition, the mutant was more sensitive to oxidative stress conferred by diamide. Using a competitive-infection assay in the murine infection model, we demonstrated for the first time that MntE is involved in the virulence of S. suis 2. Collectively, our data indicate that manganese homeostasis controlled by the manganese efflux system MntE is important for the pathogenesis of S. suis 2.

Auxotrophic Actinobacillus pleurpneumoniae grows in multispecies biofilms without the need for nicotinamide-adenine dinucleotide (NAD) supplementation.

BACKGROUND: Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumonia, which causes important worldwide economic losses in the swine industry. Several respiratory tract infections are associated with biofilm formation, and A. pleuropneumoniae has the ability to form biofilms in vitro. Biofilms are structured communities of bacterial cells enclosed in a self-produced polymer matrix that are attached to an abiotic or biotic surface. Virtually all bacteria can grow as a biofilm, and multi-species biofilms are the most common form of microbial growth in nature. The goal of this study was to determine the ability of A. pleuropneumoniae to form multi-species biofilms with other bacteria frequently founded in pig farms, in the absence of pyridine compounds (nicotinamide mononucleotide [NMN], nicotinamide riboside [NR] or nicotinamide adenine dinucleotide [NAD]) that are essential for the growth of A. pleuropneumoniae. RESULTS: For the biofilm assay, strain 719, a field isolate of A. pleuropneumoniae serovar 1, was mixed with swine isolates of Streptococcus suis, Bordetella bronchiseptica, Pasteurella multocida, Staphylococcus aureus or Escherichia coli, and deposited in 96-well microtiter plates. Based on the CFU results, A. pleuropneumoniae was able to grow with every species tested in the absence of pyridine compounds in the culture media. Interestingly, A. pleuropneumoniae was also able to form strong biofilms when mixed with S. suis, B. bronchiseptica or S. aureus. In the presence of E. coli, A. pleuropneumoniae only formed a weak biofilm. The live and dead populations, and the matrix composition of multi-species biofilms were also characterized using fluorescent markers and enzyme treatments. The results indicated that poly-N-acetyl-glucosamine remains the primary component responsible for the biofilm structure. CONCLUSIONS: In conclusion, A. pleuropneumoniae apparently is able to satisfy the requirement of pyridine compounds through of other swine pathogens by cross-feeding, which enables A. pleuropneumoniae to grow and form multi-species biofilms.

The arginine-ornithine antiporter ArcD contributes to biological fitness of Streptococcus suis.

The arginine-ornithine antiporter (ArcD) is part of the Arginine Deiminase System (ADS), a catabolic, energy-providing pathway found in a variety of different bacterial species, including the porcine zoonotic pathogen Streptococcus suis. The ADS has recently been shown to play a role in the pathogenicity of S. suis, in particular in its survival in host cells. The contribution of arginine and arginine transport mediated by ArcD, however, has yet to be clarified. In the present study, we showed by experiments using [U-(13)C6]arginine as a tracer molecule that S. suis is auxotrophic for arginine and that bacterial growth depends on the uptake of extracellular arginine. To further study the role of ArcD in arginine metabolism, we generated an arcD-specific mutant strain and characterized its growth compared to the wild-type (WT) strain, a virulent serotype 2 strain. The mutant strain showed a markedly reduced growth in chemically defined media supplemented with arginine when compared to the WT strain, suggesting that ArcD promotes arginine uptake. To further evaluate the in vivo relevance of ArcD, we studied the intracellular bacterial survival of the arcD mutant strain in an epithelial cell culture infection model. The mutant strain was substantially attenuated, and its reduced intracellular survival rate correlated with a lower ability to neutralize the acidified environment. Based on these results, we propose that ArcD, by its function as an arginine-ornithine antiporter, is important for supplying arginine as substrate of the ADS and, thereby, contributes to biological fitness and virulence of S. suis in the host.

Species selectivity of new siderophore-drug conjugates that use specific iron uptake for entry into bacteria.

Siderophores selectively bind ferric iron and are involved in receptor-specific iron transport into bacteria. Several types of siderophores were synthesized, and growth-promoting or inhibitory activities when they were conjugated to carbacephalosporin, erythromycylamine, or nalidixic acid were investigated. Overall, 11 types of siderophores and 21 drug conjugates were tested against seven different bacterial species: Escherichia coli, Bordetella bronchiseptica, Pasteurella multocida, Pasteurella haemolytica, Streptococcus suis, Staphylococcus aureus, and Staphylococcus epidermidis. In some species, the inhibitory activities of the drug conjugates were associated with the ability of the bacteria to use the siderophore portion of the molecules for growth promotion in disc diffusion tests (0.04 mumol of conjugate or siderophore per disc). E. coli used catechol-based siderophore portions as well as hydroxamate-based tri-delta-OH-N-OH-delta-N-acetyl-L-ornithine ferric iron ligands for growth under iron-restricted conditions achieved by supplemental ethylenediamine di (O-hydroxyphenylacetic acid) (100 micrograms/ml) and was sensitive to carbacephalosporin conjugated to these siderophore types (up to a 34-mm-diameter inhibition zone). B. bronchiseptica used desferrioxamine B and an isocyanurate-based or trihydroxamate in addition to catechol-based siderophore portions for promotion but was not inhibited by beta-lactam conjugates partly because of the presence of beta-lactamase. P. multocida and P. haemolytica did not use any of the synthetic siderophores for growth promotion, and the inhibitory activities of some conjugates seemed partly linked to their ability to withhold iron from these bacteria, since individual siderophore portions showed some antibacterial effects. Individual siderophores did not promote S. suis growth in restrictive conditions, but the type of ferric iron ligands attached to beta-lactams affected inhibitory activities. The antibacterial activities of the intracellular-acting agents erythromycylamine and nalidixic acid were reduced or lost, even against S. aureus and S. epidermidis, when the agents were conjugated to siderophores. Conjugate-resistant E. coli mutants showed the absence of some iron-regulated outer membrane proteins in gel electrophoresis profiles and in specific phage or colicin sensitivity tests, implying that the drugs used outer membrane receptors of ferric complexes to get into cells.