Effect of Phytochemical Compounds on Trichomonas tenax, an Oral Protozoan.

Trichomonas tenax is an oral protozoan with an estimated global pooled prevalence of 17% in the human population.1 Observational studies have demonstrated a significant statistical correlation between oral colonization by T. tenax and the progression of periodontal disease.2 Proposed pathogenic mechanisms for this protozoan include the production of tissue-damaging enzymes, induction of apoptosis in human cells, and dysbiosis of the oral microbiome.3 In patients for whom metronidazole (MTZ) is contraindicated, phytochemicals may offer a viable alternative for controlling T. tenax. Various plant extracts have shown promising in vitro activity against other trichomonads, such as T. vaginalis and Tritrichomonas foetus, as reviewed by Friedman et al.4.

Citizen Science Informs How Missourians Interact With Ticks.

Understanding which tick species Missourians interact with and where exposures occur can help prevent and diagnose tickborne diseases. A statewide survey of ticks and tickborne pathogens was conducted using citizen science. Missourians submitted 12,819 ticks from April-December 2021. In total, 71% of ticks were Amblyomma americanum, and 89% were collected from a person or a pet. These preliminary data provide tick distributions at the county level and identify patterns of direct tick exposure to humans.

Role of SigB and Staphyloxanthin in Radiation Survival of Staphylococcus aureus.

Staphylococcus aureus is a potent human pathogen. The virulence of this bacterium depends on a multitude of factors that it produces. One such virulence factor is the golden pigment, staphyloxanthin, which has been shown to protect the bacterium from oxidative stress. Expression of the staphyloxanthin biosynthetic pathway is dependent on SigB, a global stress response regulator in S. aureus. This study investigated the role of staphyloxanthin and SigB in protection of S. aureus from radiation damage. Using stationary-phase bacterial cells, it was determined that the staphyloxanthin-deficient (crt mutant) strain was significantly sensitive to UV radiation (~ threefold), but not sensitive to X-radiation. However, a SigB-deficient S. aureus that also lacks staphyloxanthin, was significantly sensitive to both UV- and X-radiation. To confirm that protection from X-radiation was due to hydroxyl radicals, effect of 3 M glycerol, a known hydroxyl scavenger, was also investigated. Glycerol increased the survival of the S. aureus sigB mutant to the wild-type level suggesting that the X-radiation sensitivity of these mutants was due to deficiency in scavenging hydroxyl radicals. In summary, SigB is critical for protection of S. aureus cells from radiation damage.

Prevalence of Tick-Borne Pathogens in Northeast Missouri.

We evaluated Amblyomma americanum (lone star tick) and Dermacentor variabilis (American dog tick) in northeast Missouri for the presence of Borrelia, Ehrlichia, and Rickettsia bacteria and Heartland virus. We screened 436 individual adult lone star ticks (86% of all ticks collected) and infection rates were 6% for B. lonestari, 19% for E. chaffeensis, 3% for E. ewingii, 36% for R. amblyommatis, and 1% for R. montanensis. In the 189 individual American dog ticks, infection rates were 19% for E. chaffeensis, 15% for E. ewingii, 4% for R. amblyommatis, and 5% for R. montanensis. In addition, we screened 20 pools of adults and 30 pools of nymphs for the Heartland virus which was not detected. Understanding the presence and epidemiology of these causative (E. chaffeensis and E. ewingii) and suspected (B. lonestari, R. amblyommatis, and R. montanensis) agents in Missouri should increase awareness of potential tick-borne disease in the medical community.

Exposure to Ticks and their Pathogens in Northeast Missouri.

While the prevalence of human pathogens has been quantified in ticks in Adair County, Missouri, the prevalence of residents acquiring tick-borne diseases and seeking medical treatment has not. A public survey (n=109) revealed that 96% of respondents reported finding attached ticks on their person; of these, 38% developed symptoms post tick bite; of these, 55% reported consultation with a health care provider. Overall, 89% of practitioners surveyed had treated at least one patient for tick-borne disease. Rocky Mountain spotted fever and Lyme disease were the most common illnesses diagnosed, however, the only confirmed cases reported by Missouri Department of Health and Senior Services from 2013-2017 were ehrlichiosis. Results from these surveys indicate that exposure to ticks is common and ehrlichiosis infections are likely underdiagnosed while Rocky Mountain spotted fever and Lyme disease are likely overdiagnosed.

Fine Epitope Mapping of Monoclonal Antibodies to the DNA Repair Protein, RadA.

Repair of DNA damage is vital to the health and survival of all organisms. In Escherichia coli, a protein known as RadA (or Sms) participates in recombinational repair, a process that uses an undamaged DNA strand in one DNA duplex to fill a gap in a homologous DNA strand in a sister DNA duplex. In a prior report, we described the production of monoclonal antibodies (MAbs) specific for RadA. Here, we investigated the epitopes recognized by two of the antibodies, MAbs 6F5 and 2A2. Premature stop codons (ochre mutations) were introduced into the radA gene at selected sites, and the truncated RadA proteins were probed by western blotting. Deletion of as few as four amino acids (457-460) from the C-terminus of RadA significantly increased the sensitivity of E. coli to ultraviolet (UV) radiation and abolished recognition of RadA by MAb 6F5. Single alanine substitutions made between positions 443-460 also adversely affected the ability of MAb 6F5 to bind to RadA, further supporting the idea that MAb 6F5 is specific for the RadA C-terminus. An ochre mutation at position 258 abolished the recognition of RadA by MAb 2A2, whereas an ochre mutation at position 279 did not, suggesting that MAb 2A2 binds to an epitope between residues 258 and 279. MAb 2A2 recognition of RadA was destroyed by endoproteinase glu-C cleavage of RadA at position 266, and by a single alanine substitution at position 265. In a competitive enzyme-linked immunosorbent assay (ELISA), a synthetic peptide comprising residues 263-273 of RadA blocked MAb 2A2 recognition of immobilized full-length RadA by more than 97%. We infer from our results that MAb 6F5 binds to the extreme C-terminus of RadA and that MAb 2A2 is specific for an epitope within positions 263-273.

Screen for genes involved in radiation survival of Escherichia coli and construction of a reference database.

A set of 3907 single-gene knockout (Keio collection) strains of Escherichia coli K-12 was examined for strains with increased susceptibility to killing by X- or UV-radiation. After screening with a high-throughput resazurin-based assay and determining radiation survival with triplicate clonogenic assays, we identified 76 strains (and associated deleted genes) showing statistically-significant increased radiation sensitivity compared to a control strain. To determine gene novelty, we constructed a reference database comprised of genes found in nine similar studies including ours. This database contains 455 genes comprised of 103 common genes (found 2-7 times), and 352 uncommon genes (found once). Our 76 genes includes 43 common genes and 33 uncommon (potentially novel) genes, i.e., appY, atoS, betB, bglJ, clpP, cpxA, cysB, cysE, ddlA, dgkA, dppF, dusB, elfG, eutK, fadD, glnA, groL, guaB, intF, prpR, queA, rplY, seqA, sufC,yadG, yagJ, yahD, yahO, ybaK, ybfA, yfaL, yhjV, and yiaL. Of our 33 uncommon gene mutants, 4 (12%) were sensitive only to UV-radiation, 10 (30%) only to X-radiation, and 19 (58%) to both radiations. Our uncommon mutants vs. our common mutants showed more radiation specificity, i.e., 12% vs. 9% (sensitive only to UV-); 30% vs. 16% (X-) and 58% vs. 74% (both radiations). Considering just our radiation-sensitive mutants, the median UV-radiation survival (75Jm-2) for 23 uncommon mutants was 6.84E-3 compared to 1.85E-3 for 36 common mutants (P=0.025). Similarly, the average X-radiation survival for 29 uncommon mutants was 1.08E-2, compared to 6.19E-3 for 39 common mutants (P=0.010). Comparing gene functions using MultiFun terms, uncommon genes tended to show less involvement in DNA repair-relevant categories (information transfer and cell processes), but greater involvement in seven other categories. Our analysis of 455 genes suggests cell survival and DNA repair processes are more complex than previously understood, and may be compromised by deficiencies in other processes.

A ΔdinB mutation that sensitizes Escherichia coli to the lethal effects of UV- and X-radiation.

The DinB (PolIV) protein of Escherichia coli participates in several cellular functions. We investigated a dinB mutation, Δ(dinB-yafN)883(::kan) [referred to as ΔdinB883], which strongly sensitized E. coli cells to both UV- and X-radiation killing. Earlier reports indicated dinB mutations had no obvious effect on UV radiation sensitivity which we confirmed by showing that normal UV radiation sensitivity is conferred by the ΔdinB749 allele. Compared to a wild-type strain, the ΔdinB883 mutant was most sensitive (160-fold) in early to mid-logarithmic growth phase and much less sensitive (twofold) in late log or stationary phases, thus showing a growth phase-dependence for UV radiation sensitivity. This sensitizing effect of ΔdinB883 is assumed to be completely dependent upon the presence of UmuDC protein; since the ΔdinB883 mutation did not sensitize the ΔumuDC strain to UV radiation killing throughout log phase and early stationary phase growth. The DNA damage checkpoint activity of UmuDC was clearly affected by ΔdinB883 as shown by testing a umuC104 ΔdinB883 double-mutant. The sensitivities of the ΔumuDC strain and the ΔdinB883 ΔumuDC double-mutant strain were significantly greater than for the ΔdinB883 strain, suggesting that the ΔdinB883 allele only partially suppresses UmuDC activity. The ΔdinB883 mutation partially sensitized (fivefold) uvrA and uvrB strains to UV radiation, but did not sensitize a ΔrecA strain. A comparison of the DNA sequences of the ΔdinB883 allele with the sequences of the Δ(dinB-yafN)882(::kan) and ΔdinB749 alleles, which do not sensitize cells to UV radiation, revealed ΔdinB883 is likely a "gain-of-function" mutation. The ΔdinB883 allele encodes the first 54 amino acids of wild-type DinB followed by 29 predicted residues resulting from the continuation of the dinB reading frame into an adjacent insertion fragment. The resulting polypeptide is proposed to interfere directly or indirectly with UmuDC function(s) involved in protecting cells against the lethal effects of radiation.

Resazurin-based assay for screening bacteria for radiation sensitivity.

We report a simple and efficient colorimetric method to screen large numbers of bacterial strains for UV- and X-radiation sensitivity. We used reference radiation-sensitive and control strains of Escherichia coli K-12 to compare our colorimetric method to a standard clonogenic plating method. Our colorimetric method was as accurate as the standard method and was superior in terms of savings in supplies and man-hours.