Arto T. Pulliainen

Bats as Reservoir Hosts of Human Bacterial Pathogen, Bartonella mayotimonensis

Bats can be potent reservoirs of human bacterial pathogens.

Persistence of Bartonella spp. stealth pathogens: from subclinical infections to vasoproliferative tumor formation

Bartonella spp. are facultative intracellular bacteria that typically cause a long-lasting intraerythrocytic bacteremia in their mammalian reservoir hosts, thereby favoring transmission by blood-sucking arthropods. In most cases, natural reservoir host infections are subclinical and the relapsing intraerythrocytic bacteremia may last weeks, months, or even years. In this review, we will follow the infection cycle of Bartonella spp. in a reservoir host, which typically starts with an intradermal inoculation of bacteria that are superficially scratched into the skin from arthropod feces and terminates with the pathogen exit by the blood-sucking arthropod. The current knowledge of bacterial countermeasures against mammalian immune response will be presented for each critical step of the pathogenesis. The prevailing models of the still-enigmatic primary niche and the anatomical location where bacteria reside, persist, and are periodically seeded into the bloodstream to cause the typical relapsing Bartonella spp. bacteremia will also be critically discussed. The review will end up with a discussion of the ability of Bartonella spp., namely Bartonella henselae, Bartonella quintana, and Bartonella bacilliformis, to induce tumor-like vascular deformations in humans having compromised immune response such as in patients with AIDS.

Dps/Dpr ferritin‐like protein: insights into the mechanism of iron incorporation and evidence for a central role in cellular iron homeostasis in Streptococcus suis

The Dps family members constitute a distinct group of multimeric and ferritin‐like iron binding proteins (up to 500 iron atoms/12‐mer) that are widespread in eubacteria and archaea and implicated in oxidative stress resistance and virulence. Despite the wealth of structural knowledge, the mechanism of iron incorporation has remained elusive. Here, we provide evidence on Dpr of the swine and human pathogen Streptococcus suis that: (i) iron incorporation proceeds by Fe(II) binding, Fe(II) oxidation and subsequent storage as Fe(III); (ii) Fe(II) atoms enter the  12‐mer cavity through four hydrophilic pores; and (iii) Fe(II) atoms are oxidized inside the 12‐mer cavity at 12 identical inter‐subunit sites, which are structurally different but functionally equivalent to the ferroxidase centres of classical ferritins. We also provide evidence, by deleting and ectopically overexpressing Dpr, that Dpr affects cellular iron homeostasis. The key residues responsible for iron incorporation in S. suis Dpr are well conserved throughout the Dps family. A model for the iron incorporation mechanism of the Dps/Dpr ferritin‐like protein is proposed.

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340.

Identification of a novel streptococcal adhesin P (SadP) protein recognizing galactosyl-α1-4-galactose-containing glycoconjugates: convergent evolution of bacterial pathogens to binding of the same host receptor.

Background: Adhesion is a prerequisite to infectious diseases. Results: A novel streptococcal Galα1–4Gal-recognizing adhesin was identified, which has no homology to known Galα1–4Gal-recognizing proteins. Conclusion: SadP is an example of convergent evolution of adhesins to binding to the same receptor, Galα1–4Gal, abundant in glycolipids. Significance: Identification of SadP helps to understand the molecular basis of streptococcal pathogenicity. Bacterial adhesion is often a prerequisite for infection, and host cell surface carbohydrates play a major role as adhesion receptors. Streptococci are a leading cause of infectious diseases. However, only few carbohydrate-specific streptococcal adhesins are known. Streptococcus suis is an important pig pathogen and a zoonotic agent causing meningitis in pigs and humans. In this study, we have identified an adhesin that mediates the binding of S. suis to galactosyl-α1–4-galactose (Galα1–4Gal)-containing host receptors. A functionally unknown S. suis cell wall protein (SSU0253), designated here as SadP (streptococcal adhesin P), was identified using a Galα1–4Gal-containing affinity matrix and LC-ESI mass spectrometry. Although the function of the protein was not previously known, it was recently identified as an immunogenic cell wall protein in a proteomic study. Insertional inactivation of the sadP gene abolished S. suis Galα1–4Gal-dependent binding. The adhesin gene sadP was cloned and expressed in Escherichia coli. Characterization of its binding specificity showed that SadP recognizes Galα1–4Gal-oligosaccharides and binds its natural glycolipid receptor, GbO3 (CD77). The N terminus of SadP was shown to contain a Galα1-Gal-binding site and not to have apparent sequence similarity to other bacterial adhesins, including the E. coli P fimbrial adhesins, or to E. coli verotoxin or Pseudomonas aeruginosa lectin I also recognizing the same Galα1–4Gal disaccharide. The SadP and E. coli P adhesins represent a unique example of convergent evolution toward binding to the same host receptor structure.

Bartonella henselae: Subversion of vascular endothelial cell functions by translocated bacterial effector proteins

Bartonella henselae (Bh) is a worldwide distributed zoonotic pathogen. Depending on the immune status of the infected individual this bacterium can cause a wide spectrum of clinical manifestations, ranging from cat scratch disease (CSD) to bacillary angiomatosis (BA) and bacillary peliosis (BP). BA and BP are characterized by tumor-like lesions at the skin or in the inner organs, respectively. These structures display pathological sprouting of capillaries with enlarged and hyperproliferated vascular endothelial cells (ECs) that are frequently found in close association with bacteria. Here we review the cellular changes observed upon Bh infection of ECs in vitro and outline the role of the VirB type IV secretion system (T4SS) and its translocated effector proteins in the modulation of EC signalling cascades. The current model how this virulence system could contribute to the vasoproliferative activity of Bh is described.

Bacterial effector binds host cell adenylyl cyclase to potentiate Gαs-dependent cAMP production

Subversion of host organism cAMP signaling is an efficient and widespread mechanism of microbial pathogenesis. Bartonella effector protein A (BepA) of vasculotumorigenic Bartonella henselae protects the infected human endothelial cells against apoptotic stimuli by elevation of cellular cAMP levels by an as yet unknown mechanism. Here, adenylyl cyclase (AC) and the α-subunit of the AC-stimulating G protein (Gαs) were identified as potential cellular target proteins for BepA by gel-free proteomics. Results of the proteomics screen were evaluated for physical and functional interaction by: (i) a heterologous in vivo coexpression system, where human AC activity was reconstituted under the regulation of Gαs and BepA in Escherichia coli; (ii) in vitro AC assays with membrane-anchored full-length human AC and recombinant BepA and Gαs; (iii) surface plasmon resonance experiments; and (iv) an in vivo fluorescence bimolecular complementation-analysis. The data demonstrate that BepA directly binds host cell AC to potentiate the Gαs-dependent cAMP production. As opposed to the known microbial mechanisms, such as ADP ribosylation of G protein α-subunits by cholera and pertussis toxins, the fundamentally different BepA-mediated elevation of host cell cAMP concentration appears subtle and is dependent on the stimulus of a G protein-coupled receptor-released Gαs. We propose that this mechanism contributes to the persistence of Bartonella henselae in the chronically infected vascular endothelium.

Deficiency of the Rgg Regulator Promotes H2O2 Resistance, AhpCF-Mediated H2O2 Decomposition, and Virulence in Streptococcus pyogenes

Streptococcus pyogenes (group A streptococcus [GAS]), a catalase-negative gram-positive bacterium, is aerotolerant and survives H2O2 exposures that kill many catalase-positive bacteria. The molecular basis of the H2O2 resistance is poorly known. Here, we demonstrate that serotype M49 GAS lacking the Rgg regulator is more resistant to H2O2 and also decomposes more H2O2 than the parental strain. Subgenomic transcriptional profiling and genome-integrated green fluorescent protein reporters showed that a bicistronic operon, a homolog of the Streptococcus mutans ahpCF operon, is transcriptionally up-regulated in the absence of Rgg. Phenotypic assays with ahpCF operon knockouts demonstrated that the gene products decompose H2O2 and protect GAS against peroxide stress. In a murine intraperitoneal-infection model, Rgg deficiency increased the virulence of GAS, although in an ahpCF-independent manner. Rgg-mediated repression of H2O2 resistance is divergent from the previously characterized peroxide resistance repressor PerR. Moreover, Rgg-mediated repression of H2O2 resistance is inducible by cellular stresses of diverse natures--ethanol, organic hydroperoxide, and H2O2. Rgg is thus identified as a novel sensoregulator of streptococcal H2O2 resistance with potential implications for the virulence of the catalase-negative GAS.

Cat Scratch Disease Caused by Bartonella grahamii in an Immunocompromised Patient

ABSTRACT Bartonella grahamii colonizes rodents worldwide and has been detected in questing Ixodes ricinus ticks. Here, the first human B. grahamii infection confirmed by multilocus sequence typing is reported. The route of transmission and clinical picture of the patient are similar to those seen in patients with cat scratch disease, which is typically diagnosed as a Bartonella henselae infection.

Expression, purification and crystallization of Dpr, a ferritin-like protein from the Gram-positive meningitis-associated bacterium Streptococcus suis

Ferritin-like proteins form a novel family of bacterial proteins with diverse functions, such as DNA binding, iron storage and cell activation. A common structural feature of these proteins is their ability to form spherical dodecamers. Dpr is a ferritin-like protein from the Gram-positive bacterium Streptococcus suis. Full-length and truncated Dpr were expressed and purified as 6xHis-tag fusion proteins. Crystals of truncated Dpr suitable for X-ray diffraction analysis were obtained after the removal of the N-terminal affinity tag by thrombin cleavage. A complete data set to 2.3 A resolution was collected using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 104.3, b = 137.6, c = 142.1 A and 12 molecules in the asymmetric unit.