International Journal of Molecular Sciences Review Mycobacterial Virulence Factors: Surface-Exposed Lipids and Secreted Proteins Angel Ly and Jun Liu * Department of Molecular Genetics, University of Toronto, Toronto, ON M5G1M1, Canada; [email protected] * Correspondence: [email protected]; Tel.: +1-416-946-5067 Received: 29 April 2020; Accepted: 1 June 2020; Published: 2 June 2020 Abstract: The clinically important Mycobacterium tuberculosis (M. tb) and related mycobacterial pathogens use various virulence mechanisms to survive and cause disease in their hosts. Several well-established virulence factors include the surface-exposed lipids in the mycobacterial outer membrane, as well as the Esx family proteins and the Pro-Glu (PE)/ Pro-Pro-Glu (PPE) family proteins secreted by type VII secretion systems (T7SS). Five ESX T7SS exist in M. tb and three—EsxA secretion system-1 (ESX-1), ESX-3, and ESX-5—have been implicated in virulence, yet only the structures of ESX-3 and ESX-5 have been solved to date. Here, we summarize the current research on three outer membrane lipids—phthiocerol dimycocerosates, phenolic glycolipids, and sulfolipids—as well as the secretion machinery and substrates of three mycobacterial T7SS—ESX-1, ESX-3, and ESX-5. We propose a structural model of the M. tb ESX-1 system based on the latest structural findings of the ESX-3 and ESX-5 secretion apparatuses to gain insight into the transport mechanism of ESX-associated virulence factors. Keywords: Mycobacterium tuberculosis; mycobacteria; virulence factors; pathogenesis; type VII secretion systems 1. Introduction The host–pathogen relationship between humans and Mycobacterium tuberculosis (M. tb) has been evolving for approximately 50,000 to 70,000 years [1], and its persistence as an infectious pathogen has led to 10 million new cases of tuberculosis (TB) and 1.2 million TB-related deaths in 2018 [2]. The clinically important pathogen M. tb belongs to the Mycobacterium genus, which is characterized by bacteria that exhibit slow-growth, high GC genome, and a distinguishingly thick cell wall [3]. TB in humans and animals results from infection by the M. tb complex, which consists of M. tb, M. africanum, M. bovis, M. canetti, M. caprae, M. microti, M. mungi, M. orygis, M. pinnipedii and M. suricattae [4–7]. These pathogens infect and multiply in alveolar macrophages to cause pulmonary TB disease [8], but they can also lead to extrapulmonary disease when bacteria disseminate outside of the lungs, such as to the central nervous system and/or lymph nodes [9]. Another class of clinically relevant mycobacteria includes non-tuberculosis mycobacteria such as the M. avium complex [10,11], M. marinum [12,13], and M. ulcerans [14], which are opportunistic pathogens that can cause disease in immunocompromised individuals. The complexities of mycobacterial physiology, morphology, and virulence mechanisms render pathogens in the Mycobacterium genus a difficult target of study for the development of effective antimycobacterial therapies. The success of mycobacterial pathogens in causing disease involves various mechanisms that enable colonization, replication, and survival in their host, thus mycobacterial virulence factors are typically defined as bacterial genes or cellular components that enable their overall survival in the host. If a deletion or loss of any particular gene or cellular component impairs bacterial growth in the host, Int. J. Mol. Sci. 2020, 21, 3985; doi:10.3390/ijms21113985 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 14 Int. J. Mol. Sci. 2020, 21, 3985 2 of 14 host, it is considered a virulence factor. Some of these factors have been identified by genomic, biochemical, and functional analysis of M. tb and related mycobacterial pathogens, therefore several itof is these considered genes aor virulence cellular components factor. Some that of these have factors well-recognized have been identifiedroles in virulence by genomic, will biochemical,be the focus andof this functional review analysis(Figure 1 of). M. tb and related mycobacterial pathogens, therefore several of these genes or cellular components that have well-recognized roles in virulence will be the focus of this review (Figure1). glucose OM 2+ glycerol Mg PPE38 PDIM PGL PE19/PPE51 PE20/PPE35 PE external PG lipid mycolic IM ESX-1 ESX-3 ESX-5 ESX-2 ESX-4 acid arabinogalactan phospholipid EsxA/EsxB EsxG/EsxH PE/PPE EspA/EspC PE5/PPE4 EspB Figure 1. Virulence factors of mycobacteria. Three type VII secretion systems—ESAT-6 secretion system-1Figure 1. (ESX-1), Virulence ESX-3, factors and of ESX-5—secrete mycobacteria. proteins Three type across VII the secretion IM. Pro-Glu systems (PE)—/Pro-Pro-GluESAT-6 secretion (PPE) small-moleculesystem-1 (ESX- selective1), ESX-3, channels and ESX transport-5—secrete nutrients proteins and across proteins the across IM. Pro the-Glu OM. (PE Various)/Pro- mycobacterialPro-Glu (PPE) cellsmall wall-molecule lipids are selective depicted onchannels the outer transport leaflet of nutrients the OM. Innerand membraneproteins across (IM); peptidoglycan the OM. Various (PG); outermycobacterial membrane cell (OM). wall lipids are depicted on the outer leaflet of the OM. Inner membrane (IM); peptidoglycan (PG); outer membrane (OM). 2. Localization in the Outer Membrane: Surface-Exposed Lipids and PE/PPE Family Proteins 2. Localization in the Outer Membrane: Surface-Exposed Lipids and PE/PPE Family Proteins The mycobacterial cell wall is unique in that it contains a layer of arabinogalactan that is covalently linkedThe to amycobacterial large amount ofcell long-chain wall is unique (C60–C90 in) fattythat acidsit contains called mycolica layer acids.of arabinogalactan This layer of mycolic that is acidscovalently forms linked the inner to a large leaflet amount of the mycobacterialof long-chain (C outer60–C90 membrane,) fatty acids whilecalled variousmycolic complexacids. This lipids, layer includingof mycolic glycolipids, acids forms form the inner the outer leaflet leaflet, of the which mycobacter togetherial formouter amembrane, thick hydrophobic while various barrier complex that is oftenlipids, di ffiincludingcult to penetrate glycolipids, with form standard the outer approaches leaflet, [which15] (Figure together1). This form characteristic a thick hydrophobic of mycobacteria barrier isthat a contributing is often difficult factor to topenetrate the ineff withectiveness standard of manyapproaches antibiotics [15] ( againstFigure 1 this). This genus. characteristic While this of sectionmycobacte willria discuss is a contributing three outer membrane factor to the lipids ineffectiveness that have well-established of many antibiotics roles as against virulence this factors, genus. theWhile Pro-Glu this (PE)section/Pro-Pro-Glu will discuss (PPE) three family outer proteins membrane will also lipids be discussed that have since well these-established proteins roles exhibit as outervirulence membrane factors, localization, the Pro-Glu have (PE) roles/ Pro in-Pro virulence,-Glu (PPE and) family may provide proteins a will newly-identified also be discussed means since for nutrientthese proteins and/or proteinexhibit transportouter membrane across this localization, hydrophobic have barrier. roles in virulence, and may provide a newly-identified means for nutrient and/or protein transport across this hydrophobic barrier. 2.1. Phthiocerol Dimycocerosates (PDIMs) and Phenolic Glycolipids (PGLs) 2.1. Phthiocerol Dimycocerosates dimycocerosates (PDIMs) (PDIMs) and andPhenolic phenolic Glycolipids glycolipids (PGLs) (PGLs) are structurally related complexPhthiocerol lipids in dimycocerosates the mycobacterial (PDIMs) outer and membrane phenolic thatglycolipids have been (PGLs) shown are structurally to be critical related for mycobacterialcomplex lipids virulence in the (Figuremycobacterial1). PDIMs outer are methyl-branchedmembrane that have fatty acid-containingbeen shown to lipidsbe critical that are for presentmycobacterial only in virulence pathogenic (Figure mycobacteria 1). PDIMs such are asmethylM. tb-,branchedM. bovis andfattyM. acid marinum-containing[16]. lipids PDIMs that were are firstpresent implicated only in inpathogenic virulence mycobacteria using signature-tagged such as M. transposon tb, M. bovis mutagenesis and M. marinum which [16]. identified PDIMsM. were tb PDIMfirst implicated mutants that in werevirulence attenuated using insignature mice [17-tagged,18]. Subsequently, transposon PDIMsmutagenesis have been which shown identified to mediate M. tb receptor-dependentPDIM mutants that phagocytosis were attenuated of M. in tb mice[19], contribute[17,18]. Subsequently, to the cell wall PDIMs permeability have been barrier shown [20 to], andmediate protect receptor against-dependent reactive nitrogenphagocytosis intermediates of M. tb [19], in contribute activated macrophagesto the cell wall [ permeability21]. More recently, barrier it[20], was and shown protect that PDIMsagainst alsoreactive contribute nitrogen to host intermediate cell escapes andin activated necrosis [macrophages22]. As many [21]. virulence More factorsrecently, do it not was function shown independently, that PDIMs also PDIM-mediated contribute to virulencehost cell escape has been and recently necrosis demonstrated [22]. As many to Int. J. Mol. Sci. 2020, 21, 3985 3 of 14 work in concert with EsxA secretion system-1 (ESX-1), a type VII secretion system in mycobacteria. An EsxA-deficient mutant in a PDIM-deficient background of M. tb showed a decreased ability