Complete Genome Sequence of Serotype III Streptococcus agalactiae Sequence Type 17 Strain 874391

Goh

Ulett

2021

Preprint

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Zinc (Zn) is an essential trace element for normal bacterial physiology but divergently, can intoxicate bacteria at high concentrations. Here, we define the molecular systems for Zn detoxification in Streptococcus agalactiae, also known as group B Streptococcus, and examine the effects of resistance to Zn stress on virulence. We compared the growth of wild-type bacteria and mutants deleted for the Zn exporter, czcD, and response regulator, sczA, using Zn-stress conditions in vitro. Macrophage antibiotic protection assays, and a mouse model of disseminated infection were used to assess virulence. Bacterial responses to Zn stress were defined by RNA-sequencing and qRTPCR. czcD and sczA enabled S. agalactiae to survive Zn stress, with the putative CzcD efflux system activated by SczA. Additional genes activated in response to Zn stress encompassed divalent cation transporters that contribute to regulation of Mn and Fe homeostasis. In vivo, the czcD-sczA Zn-management axis supported virulence in the blood, heart, liver and bladder. Additionally, several genes not previously linked to Zn stress in any bacterium, including, most notably, arcA for arginine deamination, were shown to mediate resistance to Zn stress; representing a novel molecular mechanism of bacterial resistance to Zn stress. Thus, S. agalactiae responses to Zn stress are controlled by sczA which regulates czcD, with additional mechanisms of resistance to Zn stress supported by arcA, encoding arginine deaminase. Management of Zn stress in S. agalactiae is important for virulence and bacterial survival in the host in the context of systemic infection.

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cellular management of Zinc in group BStreptococcussupports bacterial resistance against metal intoxication and promotes disseminated infection

Goh

Ulett

2021

Preprint

Self Cite

“…Primers (Supplementary Table S1) were designed using Primer3 Plus (30, 31) to quantify transcripts using Universal SYBR Green Supermix (Bio-Rad) using a Quantstudio 6 Flex (Applied Biosystems) system in accordance with MIQE guidelines (32). Standard curves were generated using five-point serial dilutions of genomic DNA (5-fold) from WT GBS 874391 (33). Expression ratios were calculated using C T values and primer efficiencies as described elsewhere (34) using dnaN , encoding DNA polymerase III β-subunit as housekeeper.…”

Section: Methodsmentioning

confidence: 99%

“…We ran paired-end 2 × 75–bp sequencing runs to align the cDNA sequences to the reference genome. For data preprocessing and bioinformatics, STAR (version 2.7.3a) was used to align the raw RNA sequencing fastq reads to the WT GBS 874391 reference genome (33). HTSeq-count, version 0.11.1, was used to estimate transcript abundances (36).…”

Section: Methodsmentioning

confidence: 99%

Copper intoxication in group BStreptococcustriggers transcriptional activation of thecopoperon that contributes to enhanced virulence during acute infection

Goh

Gosling³

et al. 2021

Preprint

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Bacteria require Copper (Cu) as an essential trace element to support cell processes; however, excess Cu can intoxicate bacteria. Here, we characterize the cop operon in group B Streptococcus (GBS), and establish its role in evasion of Cu intoxication and the response to Cu stress on virulence. Growth of GBS mutants deficient in either the copA Cu exporter, or the copY response regulator, were severely compromised in Cu-stress conditions. GBS survival of Cu stress reflected a mechanism of CopY activation of the CopA efflux system. However, neither mutant was attenuated for intracellular survival in macrophages. Analysis of global transcriptional responses to Cu by RNA-sequencing revealed a stress signature encompassing homeostasis of multiple metals. Genes induced by Cu stress included putative metal transporters for manganese import, whereas a system for iron export was repressed. In addition, copA promoted the ability of GBS to colonize the blood, liver and spleen of mice following disseminated infection. Together, these findings show that GBS copA mediates resistance to Cu intoxication, via regulation by the Cu-sensing transcriptional repressor, copY. Cu stress responses in GBS reflect a mis-metallation transcriptional signature that heightens virulence and represents an important part of the bacteria's ability to survive in different environments.

“…The bacterial strains and plasmids used in this study are listed in Table 1. The whole-genome-sequenced hypervirulent sequence type 17 (ST-17) type strain GBS 874391 (72) or its isogenic CovR-deficient derivative strain (25) was used. Plasmid pJRS9567 was kindly provided by Timothy Barnett and June Scott, Emory University.…”

Section: Methodsmentioning

confidence: 99%

Stable Expression of Modified Green Fluorescent Protein in Group B Streptococci To Enable Visualization in Experimental Systems

Ulett

2018

Appl Environ Microbiol

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Group B streptococcus (GBS) is a Gram-positive bacterium associated with various diseases in humans and animals. Many studies have examined GBS physiology, virulence, and microbe-host interactions using diverse imaging approaches, including fluorescence microscopy. Strategies to label and visualize GBS using fluorescence biomarkers have been limited to antibody-based methods or nonspecific stains that bind DNA or protein; an effective plasmid-based system to label GBS with a fluorescence biomarker would represent a useful visualization tool. In this study, we developed and validated a green fluorescent protein (GFP)-variant-expressing plasmid, pGU2664, which can be applied as a marker to visualize GBS in experimental studies. The synthetic constitutively active CP25 promoter drives strong and stable expression of the GFPmut3 biomarker in GBS strains carrying pGU2664. GBS maintains GFPmut3 activity at different phases of growth. The application of fluorescence polarization enables easy discrimination of GBS GFPmut3 activity from the autofluorescence of culture media commonly used to grow GBS. Differential interference contrast microscopy, in combination with epifluorescence microscopy to detect GFPmut3 in GBS, enabled visualization of bacterial attachment to live human epithelial cells in real time. Plasmid pGU2664 was also used to visualize phenotypic differences in the adherence of wild-type GBS and an isogenic gene-deficient mutant strain lacking CovR (the ontrolf irulenceegulator) in adhesion assays. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications. We discuss the advantages and consider the limitations of this fluorescent biomarker system developed for GBS. Group B streptococcus (GBS) is a bacterium associated with various diseases in humans and animals. This study describes the development of a strategy to label and visualize GBS using a fluorescence biomarker, termed GFPmut3. We show that this biomarker can be successfully applied to track the growth of bacteria in liquid medium, and it enables the detailed visualization of GBS in the context of live human cells in real-time microscopic analysis. The system for GFPmut3 expression in GBS described in this study provides a new tool for the visualization of this organism in diverse research applications.