The impact of nutritional immunity on Group B streptococcal pathogenesis during wound infection.

Group B Streptococcus (GBS) is a Gram-positive pathobiont that can cause adverse health outcomes in neonates and vulnerable adult populations. GBS is one of the most frequently isolated bacteria from diabetic (Db) wound infections but is rarely found in the non-diabetic (nDb) wound environment. Previously, RNA sequencing of wound tissue from Db wound infections in leprdb diabetic mice showed increased expression of neutrophil factors, and genes involved in GBS metal transport such as the zinc (Zn), manganese (Mn), and putative nickel (Ni) import systems. Here, we develop a Streptozotocin-induced diabetic wound model to evaluate the pathogenesis of two invasive strains of GBS, serotypes Ia and V. We observe an increase in metal chelators such as calprotectin (CP) and lipocalin-2 during diabetic wound infections compared to nDb. We find that CP limits GBS survival in wounds of non-diabetic mice but does not impact survival in diabetic wounds. Additionally, we utilize GBS metal transporter mutants and determine that the Zn, Mn, and putative Ni transporters in GBS are dispensable in diabetic wound infection but contributed to bacterial persistence in non-diabetic animals. Collectively, these data suggest that in non-diabetic mice, functional nutritional immunity mediated by CP is effective at mitigating GBS infection, whereas in diabetic mice, the presence of CP is not sufficient to control GBS wound persistence. IMPORTANCE Diabetic wound infections are difficult to treat and often become chronic due to an impaired immune response as well as the presence of bacterial species that establish persistent infections. Group B Streptococcus (GBS) is one of the most frequently isolated bacterial species in diabetic wound infections and, as a result, is one of the leading causes of death from skin and subcutaneous infection. However, GBS is notoriously absent in non-diabetic wounds, and little is known about why this species thrives in diabetic infection. The work herein investigates how alterations in diabetic host immunity may contribute to GBS success during diabetic wound infection.

N,N-Dimethyldithiocarbamate Elicits Pneumococcal Hypersensitivity to Copper and Macrophage-Mediated Clearance.

Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a significant cause of disease burden in pediatric and elderly populations. The rise in unencapsulated disease-causing strains and antimicrobial resistance in S. pneumoniae has increased the need for developing new antimicrobial strategies. Recent work by our laboratory has identified N,N-dimethyldithiocarbamate (DMDC) as a copper-dependent antimicrobial against bacterial, fungal, and parasitic pathogens. As a bactericidal antibiotic against S. pneumoniae, DMDC's ability to work as a copper-dependent antibiotic and its ability to work in vivo warranted further investigation. Here, our group studied the mechanisms of action of DMDC under various medium and excess-metal conditions and investigated DMDC's interactions with the innate immune system in vitro and in vivo. Of note, we found that DMDC plus copper significantly increased the internal copper concentration, hydrogen peroxide stress, nitric oxide stress, and the in vitro macrophage killing efficiency and decreased capsule. Furthermore, we found that in vivo DMDC treatment increased the quantity of innate immune cells in the lung during infection. Taken together, this study provides mechanistic insights regarding DMDC's activity as an antibiotic at the host-pathogen interface.

Novel dithiocarbamate derivatives are effective copper-dependent antimicrobials against Streptococcal species.

Despite the availability of several vaccines against multiple disease-causing strains of Streptococcus pneumoniae, the rise of antimicrobial resistance and pneumococcal disease caused by strains not covered by the vaccine creates a need for developing novel antimicrobial strategies. N,N-dimethyldithiocarbamate (DMDC) was found to be a potent copper-dependent antimicrobial against several pathogens, including S. pneumoniae. Here, DMDCs efficacy against Streptococcal pathogens Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus anginosus was tested using bactericidal and inductively coupled plasma - optical emission spectrometry. After confirming DMDC as broad-spectrum streptococcal antimicrobial, DMDC was derivatized into five compounds. The derivatives' effectiveness as copper chelators using DsRed2 and as copper-dependent antimicrobials against S. pneumoniae TIGR4 and tested in bactericidal and animal models. Two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4 and further, D39 and ATCC® 6303™ _(a type 3 capsular strain). Both Compound 3 and 4 increased the pneumococcal internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. However, in an in vivo murine pneumonia model, Compound 3, but not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. These derivatives also had detrimental effects on the other streptococcal species. Collectively, derivatizing DMDC holds promise as potent bactericidal antibiotics against relevant streptococcal pathogens.

Media Matters, Examining Historical and Modern Streptococcus pneumoniae Growth Media and the Experiments They Affect.

While some bacteria can thrive for generations in minerals and salts, many require lavish nutrition and specific chemicals to survive to the point where they can be observed and researched. Although researchers once boiled and rendered animal flesh and bones to obtain a media that facilitated bacterial growth, we now have a plethora of formulations and manufacturers to provide dehydrated flavors of historical, modified, and modern media. The purpose of media has evolved from simple isolation to more measured study. However, in some instances, media formulated to aid the metabolic, nutritional, or physical properties of microbes may not be best suited for studying pathogen behavior or resilience as a function of host interactions. While there have been comparative studies on handfuls of these media in Streptococcus pneumoniae, this review focuses on describing both the historical and modern composition of common complex (Todd Hewitt and M17), semi-defined (Adams and Roe), and defined pneumococcal media (RPMI and Van de Rijn and Kessler), key components discovered/needed for cultivation/growth enhancement, and effects these different media have on bacterial phenotypes and experimental outcomes. While many researchers find the best conditions to grow and experiment on their bacteria of choice, the reasons for some researchers to use a specific medium is at best, not discussed, and at worst, arbitrary. As such, the goal of this review is to highlight the differences in pneumococcal media to encourage investigators to challenge their decisions on why they use a given medium, discuss the recipe, and explain their reasoning.

Rules of Expansion: an Updated Consensus Operator Site for the CopR-CopY Family of Bacterial Copper Exporter System Repressors.

Copper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repressor binding. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, homologs of pneumococcal CopY have been characterized to bind a 10-base consensus sequence T/GACANNTGTA known as the cop box. Using this motif, we sought to determine whether genes outside the cop operon are also regulated by the CopY repressor, which was previously shown in Lactococcus lactis We found that S. pneumoniae CopY did not bind to cop operators upstream of these candidate genes in vitro During this process, we found that the cop box sequence is necessary but not sufficient for CopY binding. Here, we propose an updated operator sequence for the S. pneumoniaecop operon to be ATTGACAAATGTAGAT binding CopY with a dissociation constant (Kd ) of ∼28 nM. We demonstrate strong cross-species interaction between some CopY proteins and CopY operators, suggesting strong evolutionary conservation. Taken together with our binding studies and bioinformatics data, we propose the consensus operator RNYKACANNYGTMRNY for the bacterial CopR-CopY copper repressor homologs.IMPORTANCE Many Gram-positive bacteria respond to copper stress by upregulating a copper export system controlled by a copper-sensitive repressor, CopR-CopY. The previous operator sequence for this family of proteins had been identified as TACANNTGTA. Here, using several recombinant proteins and mutations in various DNA fragments, we define those 10 bases as necessary but not sufficient for binding and in doing so, refine the cop operon operator to the 16-base sequence RNYKACANNTGTMRNY. Due to the sheer number of repressors that have been said to bind to the original 10 bases, including many antibiotic resistance repressors such as BlaI and MecI, we feel that this study highlights the need to reexamine many of these sites of the past and use added stringency for verifying operators in the future.

Treatment of nitric acid-, U(VI)-, and Tc(VII)-contaminated groundwater in intermediate-scale physical models of an in situ biobarrier.

Metal and hydrogen ion acidity and extreme nitrate concentrations at Department of Energy legacywaste sites pose challenges for successful in situ U and Tc bioimmobilization. In this study, we investigated a potential in situ biobarrier configuration designed to neutralize pH and remove nitrate and radionuclides from nitric acid-, U-, and Tc-contaminated groundwater for over 21 months. Ethanol additions to groundwater flowing through native sediment and crushed limestone effectively increased pH (from 4.7 to 6.9), promoted removal of 116 mM nitrate, increased sediment biomass, and immobilized 94% of total U. Increased groundwater pH and significant U removal was also observed in a control column that received no added ethanol. Sequential extraction and XANES analyses showed U in this sediment to be solid-associated U(VI), and EXAFS analysis results were consistent with uranyl orthophosphate (UO2)3(PO4)2.4H2O(s), which may control U solubility in this system. Ratios of respiratory ubiquinones to menaquinones and copies of dissimilatory nitrite reductase genes, nirS and nirK, were at least 1 order of magnitude greater in the ethanol-stimulated system compared to the control, indicating that ethanol addition promoted growth of a largely denitrifying microbial community. Sediment 16S rRNA gene clone libraries showed that Betaproteobacteria were dominant (89%) near the source of influent acidic groundwater, whereas members of Gamma- and Alphaproteobacteria and Bacteroidetes increased along the flow path as pH increased and nitrate concentrations decreased, indicating spatial shifts in community composition as a function of pH and nitrate concentrations. Results of this study support the utility of biobarriers for treating acidic radionuclide- and nitrate-contaminated groundwater.

Changes in microbial community composition and geochemistry during uranium and technetium bioimmobilization.

In a previous column study, we investigated the long-term impact of ethanol additions on U and Tc mobility in groundwater (M. M. Michalsen et al., Environ. Sci. Technol. 40:7048-7053, 2006). Ethanol additions stimulated iron- and sulfate-reducing conditions and significantly enhanced U and Tc removal from groundwater compared to an identical column that received no ethanol additions (control). Here we present the results of a combined signature lipid and nucleic acid-based microbial community characterization in sediments collected from along the ethanol-stimulated and control column flow paths. Phospholipid fatty acid analysis showed both an increase in microbial biomass (approximately 2 orders of magnitude) and decreased ratios of cyclopropane to monoenoic precursor fatty acids in the stimulated column compared to the control, which is consistent with electron donor limitation in the control. Spatial shifts in microbial community composition were identified by PCR-denaturing gradient gel electrophoresis analysis as well as by quantitative PCR, which showed that Geobacteraceae increased significantly near the stimulated-column outlet, where soluble electron acceptors were largely depleted. Clone libraries of 16S rRNA genes from selected flow path locations in the stimulated column showed that Proteobacteria were dominant near the inlet (46 to 52%), while members of candidate division OP11 were dominant near the outlet (67%). Redundancy analysis revealed a highly significant difference (P = 0.0003) between microbial community compositions within stimulated and control sediments, with geochemical variables explaining 68% of the variance in community composition on the first two canonical axes.