antibodies Review Antibodies Inhibiting the Type III Secretion System of Gram-Negative Pathogenic Bacteria Julia A. Hotinger and Aaron E. May * Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23219, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-804-828-7134 Received: 10 June 2020; Accepted: 22 July 2020; Published: 27 July 2020 Abstract: Pathogenic bacteria are a global health threat, with over 2 million infections caused by Gram-negative bacteria every year in the United States. This problem is exacerbated by the increase in resistance to common antibiotics that are routinely used to treat these infections, creating an urgent need for innovative ways to treat and prevent virulence caused by these pathogens. Many Gram-negative pathogenic bacteria use a type III secretion system (T3SS) to inject toxins and other effector proteins directly into host cells. The T3SS has become a popular anti-virulence target because it is required for pathogenesis and knockouts have attenuated virulence. It is also not required for survival, which should result in less selective pressure for resistance formation against T3SS inhibitors. In this review, we will highlight selected examples of direct antibody immunizations and the use of antibodies in immunotherapy treatments that target the bacterial T3SS. These examples include antibodies targeting the T3SS of Pseudomonas aeruginosa, Yersinia pestis, Escherichia coli, Salmonella enterica, Shigella spp., and Chlamydia trachomatis. Keywords: type III secretion system; antibodies; prophylaxis; antibacterials; antibiotics 1. Introduction The type III secretion system (T3SS) is a multimeric protein complex used by many pathogenic Gram- negative bacteria to cause and maintain an infection [1]. Pathogens that use a T3SS include Chlamydia trachomatis, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Shigella spp., Vibrio cholerae, and Yersinia pestis [2]. The T3SS functions as a molecular syringe, sometimes called an injectisome that bacteria use to translocate effector proteins directly into a host cell (Figure1) [ 3]. The T3SS is comprised of three major components. First, a basal body that anchors the structure to the bacterial membrane containing an ATPase at the base that powers the secretion of proteins. Next, the needle itself acts as a tunnel that spans the extracellular space between the pathogen and host cell. Finally,there is a translocon that forms a pore in the host cell membrane [4]. Due to the small diameter of the needle, the effector proteins must be unfolded to be translocated and then are re-folded after entering the host cell [5]. These effector proteins are responsible for modifying the host cell functions in ways that are beneficial to the pathogen. This includes mechanisms such as reprogramming host machinery to allow for colonization through interference with actin and tubulin, gene expression, or cell cycle progression (Salmonella spp., Shigella spp.) [6,7]. Some pathogens even interfere with or induce programmed cell death (Yersinia spp., Pseudomonas spp.) [8,9]. Antibodies 2020, 9, 35; doi:10.3390/antib9030035 www.mdpi.com/journal/antibodies Antibodies 2020, 9, 35 2 of 23 Antibodies 2020, 9, x FOR PEER REVIEW 2 of 22 Figure 1.1. T3SS Structure and Common Targets. ModifiedModified fromfrom [[3].3]. TheThe T3SST3SS isis becomingbecoming anan importantimportant anti-virulenceanti‐virulence targettarget forfor manymany reasons.reasons. TheThe T3SST3SS isis specificspecific toto Gram-negativeGram‐negative pathogens,pathogens, meaning any interventionsinterventions targeting it shouldshould notnot aaffectffect commensalcommensal bacteriabacteria [[10].10]. Bacteria containing a nonfunctional T3SS also have attenuated virulence but areare stillstill capablecapable ofof growthgrowth [[11–15].11–15]. This lendslends toto thethe theorytheory thatthat inhibitinginhibiting thethe T3SST3SS willwill reducereduce thethe selectiveselective pressurepressure on on the the bacterial bacterial pathogen pathogen to to form form resistance, resistance, leading leading to slower to slower formation formation of resistance of resistance to T3SS to inhibitorsT3SS inhibitors [16]. Small [16]. moleculeSmall molecule inhibitors inhibitors of the T3SS of the have T3SS been have shown been to shown increase to survival increase rates survival after infectionrates after with infection otherwise with lethalotherwise doses lethal of bacterial doses of pathogens bacterial [pathogens10,17]. [10,17]. MammalianMammalian immune immune systems systems produce produce antibodies antibodies (Ab) against(Ab) against T3SS proteins T3SS proteins when natural when infection natural occursinfection [18 –occurs22]. Due [18–22]. to the highDue prevalenceto the high of prevalence infection caused of infection by bacteria caused utilizing by bacteria the T3SS, utilizing the majority the ofT3SS, humans the majority have antibodies of humans to thehave T3SS antibodies of some to pathogens the T3SS of already some pathogens in their system already [22 ].in Durand their system et al. tested[22]. Durand human colostrumet al. tested samples human for Abscolostrum against samples T3SS proteins for Abs for Salmonellaagainst T3SSspp., proteinsShigella spp., for Salmonella and E. coli includingspp., Shigella the spp., needle and tip, E. translocon,coli including and the secreted needle effectors.tip, translocon, They and found secreted that every effectors. sample They collected found containedthat every Abs sample to at collected least one contained of the aforementioned Abs to at least proteins one of andthe aforementioned 10% of the samples proteins contained and 10% Abs toof allthe 11 samples proteins contained tested [22 ].Abs When to all pregnant 11 proteins cattle weretested vaccinated [22]. When against pregnantE. coli cattlewith twowere recombinant vaccinated T3SS-relatedagainst E. coli proteins, with two EspB, recombinant and γ-intimin, T3SS the‐related Abs produced proteins, against EspB, and these γ‐ antigensintimin, wasthe passedAbs produced to their calvesagainst through these antigens breast milk was [ 23passed]. Rabinovitz to their etcalves al. showed through that breast calves milk with [23]. vaccinated Rabinovitz mothers et al. showedshowed markedlythat calves higher with survivalvaccinated rates mothers after a challengeshowed markedly of enterohemorrhagic higher survivalE. coli rates(EHEC) after than a challenge those with of sham-vaccinatedenterohemorrhagic mothers E. coli [ (EHEC)24]. than those with sham‐vaccinated mothers [24]. AntibodyAntibody recognitionrecognition cancan leadlead toto rapidrapid andand robustrobust responsesresponses byby thethe immuneimmune system,system, removingremoving thethe pathogenpathogen beforebefore anyany symptomssymptoms cancan bebe feltfelt oror seenseen inin thethe host.host. When thisthis isis thethe case,case, wewe considerconsider thethe hosthost toto bebe immuneimmune toto thethe pathogenpathogen [[25].25]. The presence of anti-T3SSanti‐T3SS AbsAbs isis enoughenough toto identifyidentify thatthat anan individualindividual hashas comecome intointo contactcontact withwith thethe pathogenpathogen withwith thethe T3SST3SS proteinprotein inin question,question, butbut notnot necessarilynecessarily thatthat theythey havehave immunity.immunity. This is becausebecause notnot allall AbsAbs havehave thethe samesame immunoprotectiveimmunoprotective propertiesproperties [26[26].]. Notwithstanding Notwithstanding this this fact, fact, the the presence presence of the of Abs the targeting Abs targeting the T3SS the and T3SS its e ffandectors its acrosseffectors multiple across bacterialmultiple speciesbacterial implies species a significantimplies a significant therapeutic therapeutic potential. Thepotential. most T3SSThe most structural T3SS componentsstructural components are not expressed are not byexpressed non-pathogenic by non‐pathogenic bacteria, allowing bacteria, for allowing the potential for the for potential enhanced for specificity.enhanced specificity. In this review, In this we willreview, cover we selected will cover examples selected of promising examples and of epromisingffective antibody-based and effective treatmentsantibody‐based and prophylactics treatments and that prophylactics target the T3SS that of target pathogenic the T3SS bacteria. of pathogenic bacteria. 2. Antibody Structure and Function 2. Antibody Structure and Function The majority of antibodies are “Y” shaped immunoglobulin (Ig) proteins that are used by the The majority of antibodies are “Y” shaped immunoglobulin (Ig) proteins that are used by the immune system to recognize antigens. They contain a variable domain on the tips on the Y and immune system to recognize antigens. They contain a variable domain on the tips on the Y and bind bind to antigens. A non-variable or constant domain on the stem of the Y binds to cellular receptors to antigens. A non‐variable or constant domain on the stem of the Y binds to cellular receptors (Figure 2A) [25]. There are five main isotypes of Abs found in humans: IgA, IgD, IgE, IgM and IgG. Antibodies 2020, 9, 35 3 of 23 (FigureAntibodies2A) 2020 [ 25, 9]., x There FOR PEER are REVIEW five main isotypes of Abs found in humans: IgA, IgD, IgE, IgM and3 of 22 IgG. IgA and IgG are the most commonly used in therapeutics [25,27,28]. IgA are found in the mucosal IgA and IgG are the most commonly used in therapeutics [25,27,28]. IgA are found in the mucosal membranesmembranes and and help