Biology subjects

Johnson, M. D. L.

Publications and source records attributed to Johnson, M. D. L..

4 recordsLinked to original sources

bioRxiv2023

Iron promotes copper entry in Streptococcus pneumoniae

Metals serve an important function at the host-pathogen interface, being used to leverage bacterial survival. To reduce bacterial viability in the host, some metals such as iron, are sequestered by the host, while others, such as copper are used to intoxicate bacteria. During infection, this serves the dual purpose of starving the bacteria of valuable resources while providing it with a toxic substance. By growing Streptococcus pneumoniae, a causative agent of multiple diseases including pneumonia, middle ear infections and sepsis, in the medium RPMI 1640 with a nanomolar concentration of iron, followed by exposure to a higher concentration of iron and copper, we observed an iron-dependent increase in copper association. This process was specific for iron and conserved in other S. pneumoniae serotypes. We performed single and double knockouts in selected iron transport systems and observed that under the same experimental conditions as wild-type strain, there was significantly less copper associated with the iron transport knockout bacteria. Taken together, we conclude that copper is inadvertently imported into the bacterial cell during iron acquisition.

microbiology↗

bioRxiv2023

Oral Streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages

Oral streptococci are the pioneer colonizer and structural architect of the complex oral biofilm. Disruption of this architectural framework causes oral dysbiosis associated with various clinical conditions, including dental caries, gingivitis, and oral cancer. Among the genus Streptococcus, S. anginosus is associated with esophageal, gastric, and pharyngeal cancer tissues, while S. mitis is correlated with oral cancer. However, no study has investigated mechanistic links between these Streptococcus species and cancer-related inflammatory responses. To explore the underlying involvement of S. anginosus and S. mitis in inflammation-associated cancer development, we investigated the innate immune response elicited by S. anginosus versus S. mitis using the RAW264.7 macrophage cell line. Compared to untreated or S. mitis infected macrophages, S. anginosus infected macrophages exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1{beta}, NOS2, and COX2, accompanied by enhanced NF-{kappa}B activation. Mitostress analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared to S. mitis. Further, macrophages infected with S. anginosus for 6h displayed upregulated aconitate decarboxylase, which catalyzes itaconate production. In contrast, no significant alterations were observed in succinate dehydrogenase that converts succinate to fumarate. At 24h, S. anginosus induced significant shifts in succinate and itaconate, emphasizing a unique macrophage metabolic profile, and an augmented inflammatory response in response to S. anginosus. This study underscores the capacity of S. anginosus to elicit a robust proinflammatory response in macrophages and opens new avenues of secretory immune metabolites in response to oral streptococci. ImportanceThe surge in head and neck cancer cases among individuals devoid of typical risk factors such as HPV infection, tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiomes influence on physiology, the oral microbiome, notably oral streptococci, a gatekeeper of systemic health, has been underappreciated in mucosal immunopathogenesis. S. anginosus, a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared to the early colonizer S. mitis as an initial ride towards understanding the impact of distinct oral Streptococcus species on the host immune response in the progression of OSCC.

microbiology↗

bioRxiv2022

Compounds derived from N,N-dimethyldithiocarbamate are effective copper-dependent antimicrobials against Streptococcus pneumoniae

N,N-dimethyldithiocarbamate (DMDC) is a potent copper-dependent antimicrobial against several pathogens, including Streptococcus pneumoniae. Despite the availability of several vaccines against multiple disease-causing strains of S. 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. We derived novel compounds from DMDC and tested their effectiveness as copper-dependent antimicrobials against S. pneumoniae through in vitro growth and killing curves. Compounds that caused a growth defect and were bactericidal in vitro were tested against other strains of S. pneumoniae and in complex with different transition metals. We found two compounds, sodium N-benzyl-N-methyldithiocarbamate and sodium N-allyl-N-methyldithiocarbamate (herein "Compound 3" and "Compound 4"), were effective against TIGR4, D39, and ATCC(R) 6303 (a type 3 capsular strain) and further increased the internal concentrations of copper to the same previously reported levels as with DMDC and copper treatment. We found that both Compound 3 and Compound 4 were bacteriostatic in combination with zinc. We tested Compound 3 and Compound 4 in vivo against a murine pneumonia model, finding that Compound 3, and not Compound 4, was effective in significantly decreasing the bacterial burden in the blood and lungs of S. pneumoniae-infected mice. We found that the combination of Compound 3 and copper made the pneumococcus more susceptible to activated macrophage mediated killing via an in vitro macrophage killing assay. Collectively, we demonstrate that derivatizing DMDC holds promise as potent bactericidal antibiotics against S. pneumoniae.

microbiology↗

bioRxiv2019

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

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 repression. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using S. pneumoniae CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal cop operon and comparing binding of recombinant CopY to this, and the operator sites found in Enterococcus hirae, we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive S. pneumoniae CopY operator sequence to be RnYKACAAATGTARnY (where \"R\" is purine, \"Y\" is pyrimidine, and \"K\" is either G or T) binding with an affinity of 28 nM. We further propose that the cop operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.

microbiology↗