Persistence of Staphylococcus aureus: Multiple Metabolic Pathways Impact the Expression of Virulence Factors in Small-Colony Variants (SCVs)

Abstract

Staphylococcus aureus is able to survive within host cells by switching its phenotype to the small-colony variant (SCV) phenotype. The emergence of SCVs is associated with the development of persistent infections, which may be both chronic and recurrent. This slow-growing subpopulation of S. aureus forms small colonies on solid-medium agar, is induced within host cells, presents a non-homogenous genetic background, has reduced expression of virulence factors and presents a variable phenotype (stable or unstable). While virtually all SCVs isolated from clinical specimens can revert to the parental state with rapid growth, the stable SCVs recovered in clinical specimens have been found to contain specific mutations in metabolic pathways. In contrast, other non-stable SCVs are originated from regulatory mechanisms involving global regulators (e.g., sigB, sarA, and agr) or other non-defined mutations. One major characteristic of SCVs was the observation that SCVs were recovered from five patients with infections that could persist for decades. In these five cases, the SCVs had defects in electron transport. This linked persistent infections with SCVs. The term "persistent infection" is a clinical term wherein bacteria remain in the host for prolonged periods of time, sometimes with recurrent infection, despite apparently active antibiotics. These terms were described in vitro where bacteria remain viable in liquid culture medium in the presence of antibiotics. These bacteria are called "persisters". While SCVs can be persisters in liquid culture, not all persisters are SCVs. One mechanism associated with the metabolically variant SCVs is the reduced production of virulence factors. SCVs have consistently shown reduced levels of RNAIII, a product of the accessory gene regulatory (agrBDCA) locus that controls a quorum-sensing system and regulates the expression of a large number of virulence genes. Reduced Agr acitivity is associated with enhanced survival of SCVs within host cells. In this review, we examine the impact of the SCVs with altered metabolic pathways on agr, and we draw distinctions with other types of SCVs that emerge within mammalian cells with prolonged infection.

Keywords: infection; metabolism; persisters; small colony variant (SCV); staphylococcus aureus.

FIGURE 1

SCV formation in phagocytic and non-phagocytic cells. S. aureus survives in higher numbers in non-phagocytic cells, such as osteoblasts and endothelial cells, than in phagocytic cells, such as macrophages. Because macrophages are professional phagocytes, they are able to eliminate intracellular bacteria very efficiently after 3 days, and no remaining bacteria are present to become SCVs. However, enhanced survival of S. aureus in osteoblasts and endothelial cells has been observed for up to 7 days. During the intracellular life stage of S. aureus, several bacterial cells switch to the SCV phenotype to resist intracellular stress conditions (unpublished data from L. Tuchscherr).

FIGURE 2

SCV formation within host cells. After the uptake of S. aureus by host cells, the two main possibilities are (a) the host cell dies (not described in this review) or (b) the bacteria survive for extended periods within the host cells. S. aureus uses host cells as a shelter to evade the immune response and the effects of antimicrobials. The intracellular environment triggers the selection of bacteria with mutations in metabolic genes. (c) However, the utilization of some antimicrobials can select for the formation of persisters that may or may not be SCVs. In this case, the SCVs express a stable or unstable phenotype. (d) Within host cells, S. aureus has to fight the effects of molecules generated by the host to eliminate the pathogen and the limited nutrient levels (stress). Under these conditions, dynamic SCVs are selected via cross-talk among global regulatory genes. Several SCVs carry specific auxotrophic phenotypes, such as the thymidine-, menadione- and hemin-auxotrophic. This subpopulation can rapidly switch to the original wild-type phenotype in response to environmental conditions (in rich medium, several dynamic SCVs switch to the wild-type phenotype). (e) The long-term survival of unstable SCVs of S. aureus under stress conditions selects for stable SCVs. This figure was created by Biorender.com.

FIGURE 3

Regulators of agr in S. aureus SCVs. Low levels of RNAIII, the effector molecule of the Agr quorum-sensing locus, are found in many SCVs. This figure shows the multiple levels of regulation wherein the levels of the positive regulators of agr are reduced and those of the negative regulators are increased. Green represents regulators activated in SCVs, and gray indicates no activation in SCVs. SarA and SarU are positive regulators of Agr and exhibit upregulated expression in some SCVs, such as menadione and hemin mutants. However, thymidine-auxotrophic SCVs show decreased SarA levels. Overall, regulation results in a decrease in Agr activation. Abbreviations; sigB: alternative sigma factor B; sarA = staphylococcal accessory regulator; ArlRS = autolysis-related locus two component regulator; CodY = GTP-sensing transcriptional pleiotropic repressor; SrrAB = staphylococcal respiratory regulator TCR; VraR = vancomycin resistance-associated regulator of TCR; RsaE = small non-coding RNA E; MgrA = multiple gene regulator A; CyoE = protoheme IX farnesyltransferase; agr = accessory gene regulator; hla = α-hemolysin gene; and psm = phenol soluble modulin gene.

FIGURE 4

ATP in prolonged survival of S. aureus. In SCVs, several pathways that reduce the activity of enzymes in the Krebs cycle have been described. ClpC is needed for the expression of aconitase, and its level is reduced in thymidine-auxotrophic SCVs. Reduced activity of the Krebs cycle decreases the activity of electron transport chain biosynthesis, which produces a reduced membrane potential (Δψ). This process affects the biosynthesis of ATP by F₀F₁ ATPase in SCVs.