Karsten R. O. Hazlett

PCR-Based Identification of Bacteria Associated with Endodontic Infections

ABSTRACT PCR primers that target the bacterial 16S rRNA genes (or the tuf gene for the genus Enterococcus) were used to identify 10 putative bacterial pathogens in root canals with necrotic pulp. In addition, the associations of these microorganisms with symptoms and a history of diabetes mellitus were investigated. Microbial samples from the root canals of 24 teeth with necrotic pulp were included in the study. PCR with universal bacterial primers identified bacterial DNA in 22 specimens; the remaining 2 specimens were from intact teeth that had been traumatized 6 months prior to treatment. PCR with specific primers showed that preoperative symptoms were significantly associated with the presence of Streptococcus spp. (P < 0.001 by chi-square analysis). There was also a nonsignificant trend for symptoms to be associated with Fusobacterium nucleatum and Porphyromonas gingivalis (odds ratio, >2) and for diabetes mellitus to be associated with P. gingivalis and Porphyromonas endodontalis (odds ratio, >2). Cloning and sequencing of the universal PCR product in one specimen revealed the presence of an organism related to the genus Olsenella, which has not previously been described in endodontic infections.

RpoS is not central to the general stress response in Borrelia burgdorferi but does control expression of one or more essential virulence determinants.

ABSTRACT Borrelia burgdorferi, the Lyme disease spirochete, undergoes dramatic changes in antigenic composition as it cycles between its arthropod and mammalian hosts. A growing body of evidence suggests that these changes reflect, at least in part, the need for spirochetes to adapt to the physiological stresses imposed by abrupt changes in environmental conditions and nutrient availability. In many microorganisms, global responses are mediated by master regulators such as alternative sigma factors, with Escherichia coli RpoS (σS) serving as a prototype. The importance of this transcriptional activator in other bacteria, coupled with the report by Hübner et al. (A. Hübner, X. Yang, D. M. Nolen, T. G. Popova, F. C. Cabello, and M. V. Norgard, Proc. Natl. Acad. Sci. USA 98:12724-12729, 2001) demonstrating that the borrelial RpoS ortholog controls expression of OspC and decorin-binding protein A (DbpA), prompted us to examine more closely the roles of RpoS-dependent and -independent differential gene expression in physiological adaptation by the Lyme disease spirochete. We observed that B. burgdorferi rpoS (rpoSBb) was induced following temperature shift and transcript levels were further enhanced by reduced pH (pH 6.8). Using quantitative real-time reverse transcription-PCR (RT-PCR), we demonstrated that, in contrast to its ortholog (rpoSEc) in Escherichia coli, rpoSBb was expressed at significant levels in B. burgdorferi throughout all phases of growth following temperature shift. By comparing a B. burgdorferi strain 297 rpoSBb mutant to its wild-type counterpart, we determined that RpoSBb was not required for survival following exposure to a wide range of environmental stresses (i.e., temperature shift, serum starvation, increased osmolality, reactive oxygen intermediates, and increased or reduced oxygen tension), although the mutant was more sensitive to extremes of pH. While B. burgdorferi strains lacking RpoS were able to survive within intraperitoneal dialysis membrane chambers at a level equivalent to that of the wild type, they were avirulent in mice. Lastly, RT-PCR analysis of the ospE-ospF-elp paralogous lipoprotein families complements earlier findings that many temperature-inducible borrelial loci are controlled in an RpoSBb-independent manner. Together, these data point to fundamental differences between the role(s) of RpoS in B. burgdorferi and that in E. coli. Rather than functioning as a master regulator, RpoSBb appears to serve as a stress-responsive activator of a subset of virulence determinants that, together with the RpoS-independent, differentially expressed regulon, encompass the spirochetes genetic programs required for mammalian host adaptation.

The immune response to infection with Treponema pallidum, the stealth pathogen

Cutaneous immunobiology and spirochetal molecular biology have allowed investigators to propose a conceptual framework for the development of both the innate and adaptive immune response to Treponema pallidum infection. While some clinical manifestations can be attributed to humoral responses, most can be attributed to a combination of local innate and adaptive cellular immunity.

Unique structural modifications are present in the lipopolysaccharide from colistin-resistant strains of Acinetobacter baumannii.

ABSTRACT Acinetobacter baumannii is a nosocomial opportunistic pathogen that can cause severe infections, including hospital-acquired pneumonia, wound infections, and sepsis. Multidrug-resistant (MDR) strains are prevalent, further complicating patient treatment. Due to the increase in MDR strains, the cationic antimicrobial peptide colistin has been used to treat A. baumannii infections. Colistin-resistant strains of A. baumannii with alterations to the lipid A component of lipopolysaccharide (LPS) have been reported; specifically, the lipid A structure was shown to be hepta-acylated with a phosphoethanolamine (pEtN) modification present on one of the terminal phosphate residues. Using a tandem mass spectrometry platform, we provide definitive evidence that the lipid A isolated from colistin-resistant A. baumannii MAC204 LPS contains a novel structure corresponding to a diphosphoryl hepta-acylated lipid A structure with both pEtN and galactosamine (GalN) modifications. To correlate our structural studies with clinically relevant samples, we characterized colistin-susceptible and -resistant isolates obtained from patients. These results demonstrated that the clinical colistin-resistant isolate had the same pEtN and GalN modifications as those seen in the laboratory-adapted A. baumannii strain MAC204. In summary, this work has shown complete structure characterization including the accurate assignment of acylation, phosphorylation, and glycosylation of lipid A from A. baumannii, which are important for resistance to colistin.

Adaptation of Francisella tularensis to the Mammalian Environment Is Governed by Cues Which Can Be Mimicked In Vitro

ABSTRACT The intracellular bacterium Francisella tularensis survives in mammals, arthropods, and freshwater amoeba. It was previously established that the conventional media used for in vitro propagation of this microbe do not yield bacteria that mimic those harvested from infected mammals; whether these in vitro-cultivated bacteria resemble arthropod- or amoeba-adapted Francisella is unknown. As a foundation for our goal of identifying F. tularensis outer membrane proteins which are expressed during mammalian infection, we first sought to identify in vitro cultivation conditions that induce the bacteriums infection-derived phenotype. We compared Francisella LVS grown in brain heart infusion broth (BHI; a standard microbiological medium rarely used in Francisella research) to that grown in Mueller-Hinton broth (MHB; the most widely used F. tularensis medium, used here as a negative control) and macrophages (a natural host cell, used here as a positive control). BHI- and macrophage-grown F. tularensis cells showed similar expression of MglA-dependent and MglA-independent proteins; expression of the MglA-dependent proteins was repressed by the supraphysiological levels of free amino acids present in MHB. We observed that during macrophage infection, protein expression by intracellular bacteria differed from that by extracellular bacteria; BHI-grown bacteria mirrored the latter, while MHB-grown bacteria resembled neither. Naïve macrophages responding to BHI- and macrophage-grown bacteria produced markedly lower levels of proinflammatory mediators than those in cells exposed to MHB-grown bacteria. In contrast to MHB-grown bacteria, BHI-grown bacteria showed minimal delay during intracellular replication. Cumulatively, our findings provide compelling evidence that growth in BHI yields bacteria which recapitulate the phenotype of Francisella organisms that have emerged from macrophages.

An Improved Vaccine for Prevention of Respiratory Tularemia Caused by Francisella tularensis SchuS4 Strain

Vaccination of mice with Francisella tularensis live vaccine strain (LVS) mutants described so far have failed to induce protection in C57BL/6 mice against challenge with the virulent strain F. tularensis SchuS4. We have previously reported that a mutant of F. tularensis LVS deficient in iron superoxide dismutase (sodB(Ft)) is hypersensitive to oxidative stress and attenuated for virulence in mice. Herein, we evaluated the efficacy of this mutant as a vaccine candidate against respiratory tularemia caused by F. tularensis SchuS4. C57BL/6 mice were vaccinated intranasally (i.n.) with the sodB(Ft) mutant and challenged i.n. with lethal doses of F. tularensis SchuS4. The level of protection against SchuS4 challenge was higher in sodB(Ft) vaccinated group as compared to the LVS vaccinated mice. sodB(Ft) vaccinated mice following SchuS4 challenge exhibited significantly reduced bacterial burden in lungs, liver and spleen, regulated production of pro-inflammatory cytokines and less severe histopathological lesions compared to the LVS vaccinated mice. The sodB(Ft) vaccination induced a potent humoral immune response and protection against SchuS4 required both CD4 and CD8 T cells in the vaccinated mice. sodB(Ft) mutants revealed upregulated levels of chaperonine proteins DnaK, GroEL and Bfr that have been shown to be important for generation of a potent immune response against Francisella infection. Collectively, this study describes an improved live vaccine candidate against respiratory tularemia that has an attenuated virulence and enhanced protective efficacy than the LVS.

Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

Background The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacteriums mammalian, extracellular phase. Methods/Findings SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host–adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice. Conclusion F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development.

Tp0453, A Concealed Outer Membrane Protein Of Treponema Pallidum, Enhances Membrane Permeability

The outer membrane of Treponema pallidum, the non-cultivable agent of venereal syphilis, contains a paucity of protein(s) which has yet to be definitively identified. In contrast, the outer membranes of gram-negative bacteria contain abundant immunogenic membrane-spanning beta-barrel proteins mainly involved in nutrient transport. The absence of orthologs of gram-negative porins and outer membrane nutrient-specific transporters in the T. pallidum genome predicts that nutrient transport across the outer membrane must differ fundamentally in T. pallidum and gram-negative bacteria. Here we describe a T. pallidum outer membrane protein (TP0453) that, in contrast to all integral outer membrane proteins of known structure, lacks extensive beta-sheet structure and does not traverse the outer membrane to become surface exposed. TP0453 is a lipoprotein with an amphiphilic polypeptide containing multiple membrane-inserting, amphipathic alpha-helices. Insertion of the recombinant, non-lipidated protein into artificial membranes results in bilayer destabilization and enhanced permeability. Our findings lead us to hypothesize that TP0453 is a novel type of bacterial outer membrane protein which may render the T. pallidum outer membrane permeable to nutrients while remaining inaccessible to antibody.

Broad specificity AhpC-like peroxiredoxin and its thioredoxin reductant in the sparse antioxidant defense system of Treponema pallidum

Little is known about the mechanisms by which Treponema pallidum (Tp), the causative agent of syphilis, copes with oxidative stress as it establishes persistent infection within its obligate human host. The Tp genomic sequence indicates that the bacterium’s antioxidant defenses do not include glutathione and are limited to just a few proteins, with only one, TP0509, offering direct defense against peroxides. Although this Tp peroxiredoxin (Prx) closely resembles AhpC-like Prxs, Tp lacks AhpF, the typical reductant for such enzymes. Functionally, TpAhpC resembles largely eukaryotic, nonAhpC typical 2-Cys Prx proteins in using thioredoxin (Trx, TP0919) as an efficient electron donor and exhibiting broad specificity toward hydroperoxide substrates. Unlike many of the eukaryotic Prxs, however, TpAhpC is relatively resistant to inactivation during turnover with hydroperoxide substrates. As is often observed in typical 2-Cys Prxs, TpAhpC undergoes redox-sensitive oligomer formation. Quantitative immunoblotting revealed that TpTrx and TpAhpC are present at very high levels (over 100 and 300 μM, respectively) in treponemes infecting rabbit testes; their redox potentials, at -242 ± 1 and -192 ± 2 mV, respectively, are consistent with the role of TpTrx as the cellular reductant of TpAhpC. Transcriptional analysis of select antioxidant genes confirmed the presence of high mRNA levels for ahpC and trx which diminish greatly when spirochetes replicate under in vitro growth conditions. Thus, T. pallidum has evolved an extraordinarily robust, broad-spectrum AhpC as its sole mechanism for peroxide defense to combat this significant threat to treponemal growth and survival during infection.

Discordant Results Obtained with Francisella tularensis during In Vitro and In Vivo Immunological Studies Are Attributable to Compromised Bacterial Structural Integrity

Francisella tularensis (Ft) is a highly infectious intracellular pathogen and the causative agent of tularemia. Because Ft can be dispersed via small droplet-aerosols and has a very low infectious dose it is characterized as a category A Select Agent of biological warfare. Respiratory infection with the attenuated Live Vaccine Strain (LVS) and the highly virulent SchuS4 strain of Ft engenders intense peribronchiolar and perivascular inflammation, but fails to elicit select pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ) within the first ∼72 h. This in vivo finding is discordant with the principally TH1-oriented response to Ft frequently observed in cell-based studies wherein the aforementioned cytokines are produced. An often overlooked confounding factor in the interpretation of experimental results is the influence of environmental cues on the bacteriums capacity to elicit certain host responses. Herein, we reveal that adaptation of Ft to its mammalian host imparts an inability to elicit select pro-inflammatory mediators throughout the course of infection. Furthermore, in vitro findings that non-host adapted Ft elicits such a response from host cells reflect aberrant recognition of the DNA of structurally-compromised bacteria by AIM2-dependent and -independent host cell cytosolic DNA sensors. Growth of Ft in Muller-Hinton Broth or on Muller-Hinton-based chocolate agar plates or genetic mutation of Ft was found to compromise the structural integrity of the bacterium thus rendering it capable of aberrantly eliciting pro-inflammatory mediators (e.g., TNF, IL-1β, IL-6, IL-12, and IFN-γ). Our studies highlight the profound impact of different growth conditions on host cell response to infection and demonstrate that not all in vitro-derived findings may be relevant to tularemia pathogenesis in the mammalian host. Rational development of a vaccine and immunotherapeutics can only proceed from a foundation of knowledge based upon in vitro findings that recapitulate those observed during natural infection.