Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of Small colony variant Enterobacteriaceae

AbstractIsolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The genetic and biochemical basis of SCV emergencein vivois not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates ofEscherichia coli, Citrobacter freundii, andEnterobacter hormaechei. We confirmed the isogenic basis of SCV emergence, as there were only 4 single nucleotide variants in SCV forE. coli, 5 inC. freundii, and 8 inE. hormaechei, with respect to their NCV co-isolate. In addition, a 10.2kb chromosomal segment containing 11 genes was deleted in theE. hormaecheiSCV isolate. Intriguingly, each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved iron capture. Chemical rescue confirmed the causal role of heme biosynthesis inE. coliandC. freundiiand lipoic acid inE. hormaecheiSCV isolates. Genetic rescue restored normal growth under aerobic conditions forfesandhemLinC. freundii;hemLinE. coli; andlipAinE. hormaecheiSCV isolates. Prototrophic growth in all 3 SCVEnterobacteriaceaespecies was unaffected under anaerobic culture conditionsin vitro, illustrating how SCVs may persistin vivoby abandoning the highly energetic lifestyle in an iron-limiting environment. We propose that the selective loss of functions in oxidative respiration and iron acquisition is indicative of bacterial virulence attenuation for niche specialization and persistencein vivo.ImportanceSmall colony variant (SCV) bacteria are routinely isolated in the clinical microbiology laboratory and can be notoriously difficult to treat. Most studies of the genetic underpinnings of SCV clinical isolates have examinedStaphylococcus aureusand few have looked at how SCV emerge in Gram-negative bacteria. Here, we undertook detailed characterization of three clinical isolates of SCV inEscherichia coli, Citrobacter freundii, andEnterobacter hormaecheialong with their NCV co-isolates. Genomic sequencing revealed that each SCV had at least one coding change in genes involved in both bacterial oxidative respiration and iron capture. Chemical and genetic rescue revealed that both pathways could be responsible for the small colony variant. Each of the SCV showed no growth defect compared to NCV when incubated under anaerobic conditions, indicating a potential mechanism for SCV survivalin vivo. We hypothesize that by retreating to anaerobic environments and avoiding escalating iron competition with the host, SCV have adapted to live to see another day.

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Inter- and Intraclonal Diversity of the Pseudomonas aeruginosa Proteome Manifests within the Secretome

Journal of Bacteriology ◽

10.1128/jb.185.19.5807-5814.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5807-5814 ◽

Cited By ~ 45

Author(s):

Dirk Wehmhöner ◽

Susanne Häussler ◽

Burkhard Tümmler ◽

Lothar Jänsch ◽

Florian Bredenbruch ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Iron Acquisition ◽

Culture Conditions ◽

Secretion System ◽

Type I ◽

Protein Patterns ◽

Small Colony Variant ◽

Small Colony ◽

Species Specific

ABSTRACT The proteomes of cultured Pseudomonas aeruginosa isolates from chronically infected cystic fibrosis (CF) lungs were compared by using genetically divergent clones and isogenic morphotypes of one strain. Cellular extracts gave very similar protein patterns in two-dimensional gels, suggesting that the conserved species-specific core genome encodes proteins that are expressed under standard culture conditions in vitro. In contrast, the protein profiles of extracts of culture supernatants were dependent on the growth phase, and there were significant differences between clones. The profiles also varied within clonally related morphotypes from one CF patient, including a hyperpiliated small-colony variant. Mass spectrometry revealed that this variant overexpressed proteins secreted by the type I secretion system (including proteins involved in iron acquisition) and by the type III secretion system. Furthermore, the proteins in the supernatant extracts from the small-colony variant which were recognized by sera from different CF patients varied greatly. We concluded that the secretome expression is a sensitive measure of P. aeruginosa strain variation.

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Hemin-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

Journal of Bacteriology ◽

10.1128/jb.00458-21 ◽

2021 ◽

Author(s):

Izabela Z. Batko ◽

Ronald S. Flannagan ◽

Veronica Guariglia-Oropeza ◽

Jessica R. Sheldon ◽

David E. Heinrichs

Keyword(s):

Iron Acquisition ◽

Recurrent Infection ◽

Nutritional Requirement ◽

Iron Starvation ◽

Small Colony Variants ◽

Atp Production ◽

Small Colony ◽

Sense And Respond

Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they acquire iron, yet how SCVs obtain iron remains unknown. To address this, we created a stable hemB mutant in S. aureus USA300 strain LAC. The hemB SCV utilized exogenously supplied hemin but was attenuated for growth under conditions of iron starvation. RNA-seq showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrated limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo , as administration of Desferal failed to promote hemB bacterial growth in every organ analyzed except for the kidneys. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased hemin availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that hemin is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes. Importance S. aureus small colony variants (SCVs) are associated with chronic recurrent infection and worsened clinical outcome. SCVs persist within the host despite administration of antibiotics. This study yields insight into how S. aureus SCVs acquire iron which, during infection of a host, is a difficult-to-acquire metal nutrient. Under hemin-limited conditions, hemB S. aureus is impaired for siderophore-dependent growth and, in agreement, murine infection indicates that hemin-deficient SCVs meet their nutritional requirement for iron through utilization of hemin. Importantly, we demonstrate that hemB SCVs rely upon hemin as a nutrient to promote siderophore utilization. Therefore, perturbation of heme biosynthesis and/or utilization represents a viable to strategy to mitigate the ability of SCV bacteria to acquire siderophore-bound iron during infection.

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Intra- and Extracellular Activities of Dicloxacillin and Linezolid against a ClinicalStaphylococcus aureusStrain with a Small-Colony-Variant Phenotype in anIn VitroModel of THP-1 Macrophages and anIn VivoMouse Peritonitis Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00205-10 ◽

2011 ◽

Vol 55 (4) ◽

pp. 1443-1452 ◽

Cited By ~ 14

Author(s):

Anne Sandberg ◽

Sandrine Lemaire ◽

Françoise Van Bambeke ◽

Paul M. Tulkens ◽

Diarmaid Hughes ◽

...

Keyword(s):

Cell Line ◽

Recurrent Infection ◽

Line Model ◽

Small Colony Variant ◽

Cell Line Model ◽

Optimal Dosing ◽

Small Colony ◽

Peritonitis Model

ABSTRACTThe small-colony-variant (SCV) phenotype ofStaphylococcus aureushas been associated with difficult-to-treat infections, reduced antimicrobial susceptibility, and intracellular persistence. This study represents a detailed intra- and extracellular investigation of a clinical wild-type (WT)S. aureusstrain and its counterpart with an SCV phenotype bothin vitroandin vivo, using the THP-1 cell line model and the mouse peritonitis model, respectively. Bacteria of both phenotypes infected the mouse peritoneum intra- and extracellularly. The SCV phenotype was less virulent and showed distinct bacterial clearance, a reduced multiplication capacity, and a reduced internalization ability. However, some of the SCV-infected mice were still culture positive up to 96 h postinfection, and bacteria of this phenotype could spread to the mouse kidney and furthermore revert to the more virulent WT phenotype in both the mouse peritoneum and kidney. The SCV phenotype is therefore, despite reduced virulence, an important player inS. aureuspathogenesis. In the THP-1 cell line model, both dicloxacillin (DCX) and linezolid (LZD) reduced the intracellular inocula of bacteria of both phenotypes by approximately 1 to 1.5 log10in vitro, while DCX was considerably more effective against extracellular bacteria. In the mouse peritonitis model, DCX and LZD were also able to control both intra- and extracellular infections caused by either phenotype. Treatment with a single dose of DCX and LZD was, however, insufficient to clear the SCVs in the kidneys, and the risk of recurrent infection remained. This stresses the importance of an optimal dosing of the antibiotic when SCVs are present.

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Macrophage Class A Scavenger Receptor-Mediated Phagocytosis of Escherichia coli: Role of Cell Heterogeneity, Microbial Strain, and Culture Conditions In Vitro

Infection and Immunity ◽

10.1128/iai.68.4.1953-1963.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 1953-1963 ◽

Cited By ~ 160

Author(s):

Leanne Peiser ◽

Peter J. Gough ◽

Tatsuhiko Kodama ◽

Siamon Gordon

Keyword(s):

Escherichia Coli ◽

Host Defense ◽

Bacterial Strain ◽

Chinese Hamster Ovary Cells ◽

Culture Conditions ◽

E Coli ◽

Class A

ABSTRACT Macrophage class A scavenger receptors (SR-AI and SR-AII) contribute to host defense by binding polyanionic ligands such as lipopolysaccharide and lipoteichoic acid. SR-A knockout (SR-A−/−) mice are more susceptible to endotoxic shock and Listeria monocytogenes infection in vivo, possibly due to decreased clearance of lipopolysaccharide and microorganisms, respectively. We have used flow cytometry to analyze the role of SR-A and other scavenger-like receptors in phagocytosis of bacteria in vitro. Chinese hamster ovary cells stably transfected with human SR-A bound Escherichia coli and Staphylococcus aureus but ingested few organisms. Primary human monocyte-derived macrophages (Mφ) bound and ingested E. coli more efficiently, and this was partially but selectively blocked by the general SR inhibitor, poly(I). A specific and selective role for SR-A was shown, since bone marrow culture-derived Mφ from SR-A−/− mice ingested fewer E. coli organisms than did wild-type cells, while uptake of antibody-opsonized E. coli was unaffected. SR-A-dependent uptake of E. colivaried with the bacterial strain; ingestion of DH5α and K1 by SR-A−/− Mφ was reduced by 30 to 60% and 70 to 75%, respectively. Phagocytosis and endocytosis via SR-A were markedly down-modulated when Mφ were plated on serum-coated tissue culture plastic compared to bacteriologic plastic, where cell adhesion is mediated by SR-A and CR3, respectively. This paper demonstrates that SR-A can bind and ingest bacteria directly, consistent with a role in host defense in vivo, and highlights the importance of the source of the Mφ, bacterial strain, and culture conditions on receptor function in vitro.

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In Vitro and In Vivo Characterization of a Small-Colony Variant of the Primary Form of Photorhabdus luminescensMD (Enterobacteriaceae)

Applied and Environmental Microbiology ◽

10.1128/aem.64.9.3214-3219.1998 ◽

1998 ◽

Vol 64 (9) ◽

pp. 3214-3219 ◽

Cited By ~ 10

Author(s):

Kaiji Hu ◽

John M. Webster

Keyword(s):

Galleria Mellonella ◽

Photorhabdus Luminescens ◽

Small Colony Variant ◽

Small Colony ◽

High Level ◽

In Vivo Characterization ◽

Primary Form

ABSTRACT A small-colony variant (Vsm) of the primary form (Vp) ofPhotorhabdus luminescens MD from in vitro and in vivo cultures is described. Unlike the primary form, Vp, the Vsm variant is not the preferred diet of its nematode symbiont, aHeterorhabditis sp., does not support development and reproduction of the nematode, and is less pathogenic than Vp toGalleria mellonella larvae. Vsm cells were carried by 25% of infective juveniles, but they comprised a very low percentage (∼0.4%) of the total cells carried by the juvenile. In vitro subculture and in vivo injection into the larvae with either Vp or Vsm always produced a mixture of both Vp and Vsm. In nematode-bacterium-infected G. mellonella larvae, the Vp population in the hemocoel was high (4 × 109 to 5 × 109 CFU/g of wet insect tissue) at 24 h after infection, decreased about 10-fold by 48 h, and then regained a high level at day 5 before decreasing at day 7 and then remaining relatively constant through day 15 postinfection. The Vsm population, under the same conditions as those of Vp, increased gradually to a high level (9 × 108 CFU/g of wet insect tissue) at day 5 postinfection and then declined gradually through day 15.

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Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate

Applied and Environmental Microbiology ◽

10.1128/aem.02667-08 ◽

2009 ◽

Vol 75 (10) ◽

pp. 3137-3145 ◽

Cited By ~ 79

Author(s):

Hsien-Chung Tseng ◽

Collin H. Martin ◽

David R. Nielsen ◽

Kristala L. Jones Prather

Keyword(s):

Escherichia Coli ◽

Shake Flask ◽

Maximal Activity ◽

Culture Conditions ◽

Recombinant Enzymes ◽

E Coli ◽

Enhanced Production ◽

K 12

ABSTRACT Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter−1 and 2.08 g liter−1, respectively, in shake flask cultures within 2 days. The NADPH/NADP+ ratio was found to be two- to three-fold higher than the NADH/NAD+ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

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Heme-dependent siderophore utilization promotes iron-restricted growth of the Staphylococcus aureus hemB small colony variant

10.1101/2021.06.29.450315 ◽

2021 ◽

Author(s):

Izabela Z Batko ◽

Ronald S Flannagan ◽

Veronica Guariglia-Oropeza ◽

Jessica R Sheldon ◽

David E Heinrichs

Keyword(s):

Staphylococcus Aureus ◽

Iron Acquisition ◽

Iron Starvation ◽

Minimal Growth ◽

Atp Production ◽

Small Colony ◽

Sense And Respond ◽

Outstanding Question

The ability to acquire iron is essential for Staphylococcus aureus to cause infection. Respiration deficient S. aureus small colony variants (SCVs) frequently cause persistent infections, which necessitates they too acquire iron. How SCVs obtain iron remains unknown and so here we addressed this outstanding question by creating a stable hemB mutant in S. aureus USA300 strain LAC. The mutant, auxotrophic for hemin, was assessed for its ability to grow under iron-restriction and with various iron sources. The hemB SCV utilizes exogenously supplied heme but was attenuated for growth under conditions of iron starvation. RNA-seq analyses showed that both WT S. aureus and the hemB mutant sense and respond to iron starvation, however, growth assays show that the hemB mutant is defective for siderophore-mediated iron acquisition. Indeed, the hemB SCV demonstrates limited utilization of endogenous staphyloferrin B or exogenously provided staphyloferrin A, Desferal, and epinephrine, which enabled the SCV to sustain only minimal growth in iron deplete media. Direct measurement of intracellular ATP in hemB and WT S. aureus revealed that both strains can generate comparable levels of ATP during exponential growth suggesting defects in ATP production cannot account for the inability to efficiently utilize siderophores. Defective siderophore utilization by hemB bacteria was also evident in vivo. Indeed, the administration of Desferal failed to promote hemB bacterial growth in vivo, in contrast to WT, in every organ analyzed except for the murine kidney where growth was enhanced. In support of the hypothesis that S. aureus accesses heme in kidney abscesses, in vitro analyses revealed that increased heme availability enables hemB bacteria to utilize siderophores for growth when iron availability is restricted. Taken together, our data support the conclusion that heme is not only used as an iron source itself, but as a nutrient that promotes utilization of siderophore-iron complexes.

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Extracellular DNA released by glycine-auxotrophic Staphylococcus epidermidis small colony variant facilitates catheter-related infections

Communications Biology ◽

10.1038/s42003-021-02423-4 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Junlan Liu ◽

Zhen Shen ◽

Jin Tang ◽

Qian Huang ◽

Ying Jian ◽

...

Keyword(s):

Staphylococcus Epidermidis ◽

Biofilm Formation ◽

Underlying Mechanism ◽

Extracellular Dna ◽

Small Colony Variant ◽

Persistent Infections ◽

Small Colony

AbstractThough a definitive link between small colony variants (SCVs) and implant-related staphylococcal infections has been well-established, the specific underlying mechanism remains an ill-explored field. The present study analyzes the role SCVs play in catheter infection by performing genomic and metabolic analyses, as well as analyzing biofilm formation and impacts of glycine on growth and peptidoglycan-linking rate, on a clinically typical Staphylococcus epidermidis case harboring stable SCV, normal counterpart (NC) and nonstable SCV. Our findings reveal that S. epidermidis stable SCV carries mutations involved in various metabolic processes. Metabolome analyses demonstrate that two biosynthetic pathways are apparently disturbed in SCV. One is glycine biosynthesis, which contributes to remarkable glycine shortage, and supplementation of glycine restores growth and peptidoglycan-linking rate of SCV. The other is overflow of pyruvic acid and acetyl-CoA, leading to excessive acetate. SCV demonstrates higher biofilm-forming ability due to rapid autolysis and subsequent eDNA release. Despite a remarkable decline in cell viability, SCV can facilitate in vitro biofilm formation and in vivo survival of NC when co-infected with its normal counterparts. This work illustrates an intriguing strategy utilized by a glycine-auxotrophic clinical S. epidermidis SCV isolate to facilitate biofilm-related infections, and casts a new light on the role of SCV in persistent infections.

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Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

10.26434/chemrxiv.7725002.v1 ◽

2019 ◽

Author(s):

Priya Prakash ◽

Travis Lantz ◽

Krupal P. Jethava ◽

Gaurav Chopra

Keyword(s):

Amyloid Beta ◽

Western Blots ◽

Force Microscopy ◽

E Coli ◽

Atomic Force ◽

Set Up ◽

Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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