No abstract

Staphylococcus aureus is an important pathogen of humans and animals, and antibiotic resistance is a public health concern. Biofilm formation is essential in virulence and pathogenesis, and the ability to resist antibiotic treatment results in difficult-to-treat and persistent infections. As such, novel antimicrobial approaches are of great interest to the scientific, medical, and agriculture communities. We recently proposed that modulating levels of the cyclic dinucleotide signaling molecule, c-di-GMP (cyclic diguanylate [3,5-cyclic diguanylic acid], cGpGp), has utility in regulating phenotypes of prokaryotes. We report that extracellular c-di-GMP shows activity against human clinical and bovine intramammary mastitis isolates of S. aureus, including methicillinresistant S. aureus (MRSA) isolates. We show that chemically synthesized c-di-GMP is soluble and stable in water and physiological saline and stable following boiling and exposure to acid and alkali. Treatment of S. aureus with extracellular c-di-GMP inhibited cell-to-cell (intercellular) adhesive interactions in liquid medium and reduced (>50%) biofilm formation in human and bovine isolates compared to untreated controls. c-di-GMP inhibited the adherence of S. aureus to human epithelial HeLa cells. The cyclic nucleotide analogs cyclic GMP and cyclic AMP had a lesser inhibitory effect on biofilms, while 5-GMP had no major effect. We propose that cyclic dinucleotides such as c-di-GMP, used either alone or in combination with other antimicrobial agents, represent a novel and attractive approach in the development of intervention strategies for the prevention of biofilms and the control and treatment of infection.

SummaryEnteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL . To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant . We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast twohybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.

The gold-standard method for quantifying neutralizing antibody responses to many viruses, including dengue virus (DENV), is the plaque reduction neutralization test (PRNT, also called the immunofocus reduction neutralization test). The PRNT conducted on 96-well plates is high-throughput and requires a smaller volume of antiserum than on 6- or 24-well plates, but manual plaque counting is challenging and existing automated plaque counters are expensive or difficult to optimize. We have developed Viridot (Viridot package), a program for R with a user interface in shiny, that counts viral plaques of a variety of phenotypes, estimates neutralizing antibody titers, and performs other calculations of use to virologists. The Viridot plaque counter includes an automatic parameter identification mode (misses <10 plaques/well for 87% of diverse DENV strains [n = 1521]) and a mode that allows the user to fine-tune the parameters used for counting plaques. We compared standardized manual and Viridot plaque counting methods applied to the same wells by two analyses and found that Viridot plaque counts were as similar to the same analyst's manual count (Lin’s concordance correlation coefficient, ρc = 0.99 [95% confidence interval: 0.99–1.00]) as manual counts between analysts (ρc = 0.99 [95% CI: 0.98–0.99]). The average ratio of neutralizing antibody titers based on manual counted plaques to Viridot counted plaques was 1.05 (95% CI: 0.98–1.14), similar to the average ratio of antibody titers based on manual plaque counts by the two analysts (1.06 [95% CI: 0.84–1.34]). Across diverse DENV and ZIKV strains (n = 14), manual and Viridot plaque counts were mostly consistent (range of ρc = 0.74 to 1.00) and the average ratio of antibody titers based on manual and Viridot counted plaques was close to 1 (0.94 [0.86–1.02]). Thus, Viridot can be used for plaque counting and neutralizing antibody titer estimation of diverse DENV strains and potentially other viruses on 96-well plates as well as for formalization of plaque-counting rules for standardization across experiments and analysts.

Enteropathogenic Escherichia coli (EPEC) is an important cause of infantile diarrhea, especially in developing countries. EspB, a key virulence factor of EPEC, is required for the attaching and effacing effect characteristic of EPEC and enterohemorrhagic E. coli and has been posited to play several functions in the process of infection. Attaching and effacing activity is associated with the accumulation of filamentous actin beneath adherent bacteria as measured in the fluorescence actin staining (FAS) test. To determine whether different domains of EspB are responsible for different functions, 42 plasmids carrying mutated espB were introduced into an espB deletion mutant. Two major groups of espB mutants were identified. One group of 17 mutants exhibited positive FAS results and normal levels of hemolytic activity. Another group of 22 mutants exhibited negative FAS results and low levels of hemolytic activity. Three mutants were exceptional. One mutant was FAS positive but had significantly reduced hemolytic activity. Conversely, a second mutant was FAS negative but had full hemolytic activity. A third mutant had a significantly reduced FAS level compared to the wild type but full hemolytic activity. The results of EspF and Tir translocation assays confirmed that FAS-negative insertions disrupt effector translocation and mutants with FAS-positive insertions retain protein translocation activity. These results suggest that EspB has distinct domain functions involved in effector translocation that can be distinguished from its role as a component of the translocation pore.Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea in developing countries. The hallmark of EPEC infection is the ability of the pathogen to attach intimately to the host cell membrane, destroy microvilli, and induce the formation of cup-like pedestals composed of cytoskeletal proteins directly underneath the adherent bacteria. This phenomenon, known as the attaching and effacing (A/E) effect (38), has been observed in vitro in cell culture and in duodenal or rectal biopsy specimens from infants with EPEC infection (45, 58). A 35,624-bp genetic element known as the locus of enterocyte effacement (LEE) is necessary and sufficient for the A/E effect (35, 36). The LEE has 41 open reading frames whose products can be divided into five categories: a type III secretion system (