Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A

Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Evasion of serum antibodies and complement by Salmonella Typhi and Paratyphi A View ORCID Profile Fermin E. Guerra , Joyce E. Karlinsey , Stephen J. Libby , View ORCID Profile Ferric C. Fang doi: https://doi.org/10.1101/2025.01.20.633845 Fermin E. Guerra 1 Department of Laboratory Medicine and Pathology, University of Washington , Seattle, Washington, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Fermin E. Guerra Joyce E. Karlinsey 2 Department of Microbiology, University of Washington , Seattle, Washington, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Stephen J. Libby 1 Department of Laboratory Medicine and Pathology, University of Washington , Seattle, Washington, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ferric C. Fang 1 Department of Laboratory Medicine and Pathology, University of Washington , Seattle, Washington, United States of America 2 Department of Microbiology, University of Washington , Seattle, Washington, United States of America Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ferric C. Fang For correspondence: fcfang{at}uw.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Nontyphoidal and enteric fever serovars of Salmonella enterica display distinctive interactions with serum antibodies and the complement system, which initiate the host immune response to invading microbes. This study examines the contributions of lipopolysaccharide O-antigen and the S. Typhi Vi polysaccharide capsule to serum resistance, complement activation and deposition, and immunoglobulin (Ig) binding in nontyphoidal S. enterica serovar Typhimurium and the enteric fever serovars S. Typhi and S. Paratyphi A. Although all three serovars are resistant to serum killing, S. Typhi and S. Paratyphi A exhibit lower levels of Ig binding, complement binding and complement activation compared to S. Typhimurium. In S. Typhimurium, WzzB-dependent long O-antigen production and FepE-dependent very long O-antigen production are required for serum resistance but do not prevent IgM binding or complement deposition. S. Typhi lacks very long O-antigen, but its production of Vi capsule inhibits IgM binding and complement deposition, while acting in concert with long O-antigen to resist serum killing. In S. Paratyphi A, long O-antigen production is deficient due to a hypofunctional WzzB protein, but this is compensated by greater quantities of very long O-antigen, which are required for serum resistance. Restoration of WzzB function by exchange with the S. Typhimurium or S. Typhi wzzB alleles can restore long O-antigen production in S. Paratyphi A but decreases very long O-antigen production, resulting in increased IgM binding. Replacement of the S. Paratyphi A O2-type polysaccharide with the S. Typhi O9 polysaccharide further increases IgM binding of S. Paratyphi A, which enhances complement activation but not complement deposition. Lastly, a gene duplication of rfbV in S. Paratyphi A is necessary for higher levels of very long O-antigen and resistance to complement deposition and antibody binding. Collectively, these observations demonstrate fundamental differences between nontyphoidal and enteric fever Salmonella serovars in their interactions with innate immune effectors. Author Summary Enteric fever acquired by ingestion of food or water contaminated with Salmonella Typhi or Paratyphi A is a significant cause of morbidity and mortality in low- and middle-income countries. In this study, we dissect the different mechanisms by which these bacteria avoid binding by serum proteins that are required for the initiation of protective immune responses. This is in contrast to the nontyphoidal serovar Salmonella Typhimurium, a common cause of gastroenteritis, which does not avoid complement activation nor antibody binding but instead exploits host inflammation. Our observations provide new insights into the mechanisms responsible for the distinctive immunological features of human enteric fever and can inform the development of Salmonella vaccines that target the Salmonella cell envelope. Introduction Salmonella enterica is an enteric Gram-negative pathogen that causes disease in humans with clinical manifestations ranging from asymptomatic carriage to self-limited acute gastroenteritis or life-threatening systemic infection [ 1 ]. Antisera have long been used to subdivide Salmonella into >2,500 serovars on the basis of specific lipopolysaccharide (LPS) and flagellar signatures, although only a few of these are prominent etiologic agents of human disease with distinctive clinical features [ 2 ]. Ingestion of nontyphoidal S. enterica serovar Typhimurium ( S. Typhimurium) in contaminated food typically causes symptoms of acute gastroenteritis following a brief incubation period of 12 to 36 hours, with occasional extraintestinal complications that are particularly seen in individuals with immunosuppressive conditions [ 3 ]. In contrast, the enteric fever Salmonella serovars Typhi or Paratyphi A ( S . Typhi or S . Paratyphi A) exhibit a more prolonged incubation period, typically lasting from 7 to 14 days, followed by fever, bacteremia, and systemic illness [ 4 ]. Complications of enteric fever include gastrointestinal bleeding, intestinal perforation, and encephalopathy, which is associated with high mortality, and some infected individuals become chronic asymptomatic carriers despite antibiotic treatment [ 4 – 6 ]. Salmonellosis caused by nontyphoidal serovars account for over 1 million infections in the United States annually, while enteric fever serovars are estimated to cause between 12 and 27 million infections and 200,000 deaths globally each year [ 7 , 8 ]. Enteric fever is becoming increasingly difficult to treat due to the prevalence of multidrug-resistant strains [ 9 ]. Vaccines against S. Typhi are available but only partially protective and require booster doses, and currently there are no vaccines available for S. Paratyphi A or S. Typhimurium [ 10 , 11 ]. Decades of research have pointed to fundamental differences in the interaction of nontyphoidal and enteric fever Salmonella serovars in their interaction with the host immune system. Nontyphoidal serovars such as S. Typhimurium elicit an intense acute inflammatory response in the intestine, which is exploited by the bacterium to outcompete the commensal microbiota and facilitate transmission to other hosts by the production of inflammatory diarrhea [ 12 – 15 ]. In contrast, the enteric fever serovar S. Typhi employs a variety of mechanisms to avoid the stimulation of an acute inflammatory response, allowing it to persist within mononuclear cells and disseminate to extraintestinal sites [ 16 – 21 ]. Thus, while all Salmonella serovars share a common requirement to withstand host immune mediators, nontyphoidal and enteric fever serovars differ in whether to exploit or evade innate immunity. The ability of Salmonella to cause invasive disease and bloodstream infection requires resistance to serum bactericidal mechanisms primarily mediated by the complement system, a complex surveillance system that bridges innate and adaptive immunity through the classical pathway of complement activation [ 22 ]. The classical pathway is defined by immunoglobulin G (IgG) or M (IgM) binding to antigen, which induces a conformational change to recruit the complement protein C1q and the associated serine proteases C1r 2 s 2 . Serine proteases cleave complement proteins C4 and C2 to form the C3 convertase C4b2b, also known as C4b2a [ 23 ]. The C3 convertase is covalently bound to the bacterial surface or to the antigen recognition complex and cleaves complement protein C3 into the anaphylatoxin C3a and the C3b fragment. An internal thioester bond in C3b serves as the site of nucleophilic attack by hydroxyl and amino groups, which covalently link C3b to the bacterial surface [ 24 , 25 ]. When C3b binds to the C3 convertase C4b2b, the C5 convertase C4b2bC3b preferentially cleaves C5 into C5a and C5b. Complement fragment C5b forms a lipophilic complex with C6 and C7 to initiate insertion into targeted bacterial membranes. Sequential recruitment of C8 and C9 polymerization completes formation of the membrane attack complex (MAC), an 11 nm wide α-barrel pore on the bacterial membrane that leads to lysis and death [ 26 , 27 ]. In addition to mediating bacterial lysis, the anaphylatoxins C3a and C5a recruit inflammatory cells to the site of infection, and bound C3b promotes opsonophagocytosis of bacteria by phagocytic cells bearing complement receptors. Lipopolysaccharide chains attached to the outer leaflet of the outer membrane of Salmonella enterica are exposed to the extracellular environment and provide resistance to the lytic action of complement [ 28 – 33 ]. The lipopolysaccharide of Salmonella enterica is composed of a hydrophobic membrane-inserted lipid A domain that is attached to a hydrophilic nonrepeating inner and outer core oligosaccharide [ 28 ]. The outer core is attached to O-antigen repeating units, which in the Salmonella serovars Typhimurium, Typhi, and Paratyphi A share a common backbone composed of (→2)-D-mannose-(α1→4)-L-rhamnose-(α1→3)-D-galactose-(α1→) [ 34 ]. However, the distinct S. Typhimurium O4, S. Paratyphi A O2, and S. Typhi O9-antigens result from the (α1→3) linkage of D-abequose, D-paratose, or D-tyvelose, respectively, to D-mannose in the backbone. The extent of O-antigen repeating units attached to the lipid A-core is controlled by the chain length determinants WzzB and FepE. WzzB-dependent long O-antigen contains 16-to-35 O-antigen repeating units, while FepE-dependent very long O-antigen contains >100 repeating units [ 30 , 35 , 36 ]. In S. Typhi, fepE is a pseudogene, and the Vi capsule encoded by Salmonella pathogenicity island-7 (SPI-7) is produced rather than very long O-antigen [ 37 , 38 ]. The roles of long and very long O-antigen and the Vi capsule in serum resistance have been studied in S. Typhimurium and S. Typhi, and these structures are important vaccine targets. Previous studies have shown that WzzB-dependent long O-antigen confers resistance to serum killing in S . Typhimurium and S . Typhi [ 31 – 33 , 39 , 40 ]. The S . Typhi Vi capsule also confers resistance to serum killing, but FepE-dependent very long O-antigen is not required for serum resistance in S. Typhimurium [ 32 , 39 ]. In contrast, the roles of long and very long O-antigen in S . Paratyphi A serum resistance are less clear. Hiyoshi et al. assessed the influence of very long O-antigen on the phagocyte oxidative burst in S. Paratyphi A and suggested that it is required for serum resistance, but the contribution of long O-antigen was not assessed [ 20 ]. In the present study, we compared the contributions of long and very long O-antigen to serum resistance and complement activation in S. Typhimurium, S. Typhi, and S. Paratyphi A by measuring serum bactericidal activity, complement deposition, immunoglobulin binding, and anaphylatoxin release of wild-type and mutant Salmonella strains lacking WzzB, FepE, and/or the Vi capsular polysaccharide. Our novel results show that very long O-antigen is the major mechanism of serum resistance in S. Paratyphi A, that duplication of the rfbV glycosyltransferase gene contributes to serum resistance in S. Paratyphi A by enhancing the quantity of O-antigen production, that long O-antigen is deficient in S. Paratyphi A due to a WzzB point mutation, and that O-antigen length and composition significantly impact immunoglobulin binding and complement activation. Results Nontyphoidal and enteric fever Salmonella serovars differ in mechanisms of serum resistance and binding of complement and immunoglobulins Serum resistance and binding of complement and immunoglobulins by wild-type S. Typhimurium, S. Typhi, and S. Paratyphi A and their mutant derivatives were compared. Isogenic wzzB , fepE , and vexA mutant derivatives were constructed to eliminate production of long O-antigen, very long O-antigen (in S. Typhimurium and S. Paratyphi A only), or Vi capsule (in S. Typhi only), respectively. Bacteria were incubated with 50% human serum at 37 °C for 60 min, diluted, and plated for enumeration of viable cells. All wild-type serovars were resistant to serum killing, but the contribution of long or very long O-antigen to serum resistance was serovar-dependent ( Fig 1A ). Loss of WzzB-dependent long O-antigen production increased serum sensitivity in S. Typhimurium and S. Typhi but not in S. Paratyphi A. Loss of FepE-dependent very long O-antigen production did not significantly increase serum sensitivity in S. Typhimurium but rendered S. Paratyphi A highly susceptible to serum killing. Although S. Typhi does not produce very long O-antigen because fepE is a pseudogene, loss of Vi capsule increased sensitivity to serum killing and had an additive effect when combined with loss of long O-antigen. None of the three serovars were able to survive serum exposure in the absence of long and very long O-antigen and the Vi capsule. Download figure Open in new tab Fig 1. The contribution of O-antigen length and the Vi capsule to serum resistance is serovar dependent. (A) Serum Resistance. Wild-type S. Typhimurium, S. Typhi, and S. Paratyphi A are resistant to complement-mediated killing. S. Typhimurium depends on both long and very long O-antigen to resist complement killing, whereas serum resistance of S. Typhi is dependent on Vi capsule and long O-antigen. S. Paratyphi A requires very long O-antigen to resist serum killing, but long O-antigen is dispensable. Complement bactericidal activity was assayed in 50% human pooled serum after 60 min incubation at 37 °C. (B) and (C) C3b Deposition. Complement resistance mediated by the Vi capsule and by long and very long O-antigen in S. Typhi and S. Paratyphi A is inversely proportional to C3b deposition. However, S. Typhimurium serum resistance is not associated with reduced C3b deposition. Representative flow cytometry histograms show increased C3b deposition on S. Typhimurium compared to S. Typhi and S. Paratyphi A. (D) IgG Binding. S. Typhimurium long and very long O-antigen enhances IgG binding, whereas the S. Typhi Vi capsule prevents IgG binding. S. Paratyphi A very long O-antigen prevents IgG binding. (E) IgM Binding. Long and very long O-antigen are the primary IgM antigenic binding determinants in S. Typhimurium. The S. Typhi Vi capsule prevents IgM binding. Deficient long O-antigen in S. Paratyphi A enhances IgM binding. C3b and immunoglobulin binding were determined following 20 min incubation at 37 °C in 50% human pooled serum. Statistical analysis was performed by one-way ANOVA on 3-5 independent replicates, with P values in red indicating statistical significance with P <0.05. Column bars represent means, with error bars showing standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A; FI, fluorescence intensity. Deposition of complement component C3b is an essential step in complement-mediated serum killing [ 22 ]. Therefore, we used flow cytometry to determine the effect of O-antigen length on the deposition of C3b on the bacterial surface. S. Typhimurium exhibits higher levels of C3b deposition in comparison to S. Typhi and S. Paratyphi A ( Fig 1B and 1C ). Long and very long O-antigen have only modest effects on the percent of S. Typhimurium with C3b deposition, even though the loss of both WzzB and FepE results in increased serum killing, indicating that serum resistance in S. Typhimurium does not result from avoidance of C3b deposition. Instead, serum resistance in S. Typhimurium appears to depend on the localization of C3b deposition, as a wzzB mutant, which lacks long O-antigen, has less total C3b deposition yet is more susceptible to serum killing. Similarly, the loss of long O-antigen in S. Typhi increases serum susceptibility but does not significantly increase C3b deposition. In contrast, loss of the Vi capsule significantly increases the percent of S. Typhi with C3b deposition, corroborating previous studies of its anti-opsonic function [ 32 ]. In S. Paratyphi A, very long O-antigen was found to be the major determinant of C3b deposition as well as serum killing ( Fig 1A ). Loss of WzzB-dependent long O-antigen production was observed to result in a small but significant decrease in C3b deposition in S. Paratyphi A. Serum immunoglobulins bind antigen to catalyze complement activation, and bacteria have evolved mechanisms to inhibit antibody recognition and the alternative complement pathway [ 41 – 44 ]. We analyzed the effect of long and very long O-antigen on the binding of IgG ( Fig 1D ) and IgM ( Fig 1E ) to Salmonella . All three serovars exhibited similar levels of IgG binding. In S. Typhimurium, loss of long and very long O-antigen results in a small but significant decrease in IgG binding. The S. Typhi Vi capsule prevents IgG binding, but loss of long O-antigen does not have a significant effect on IgG binding. Similarly, loss of very long O-antigen in S. Paratyphi A increases IgG binding. The loss of long and very-long O-antigen reduces IgG binding compared to loss of very long O-antigen alone, suggesting that IgG may bind to long O-antigen in the absence of very long O-antigen in S. Paratyphi A. As with C3b deposition, higher levels of IgM binding were observed in S. Typhimurium compared to S. Typhi and S. Paratyphi A. IgM binding to S. Typhimurium is dependent on the presence of both long and very long O-antigen ( Fig 1E ). Increased IgM binding to S. Typhi occurs in the absence of Vi capsule, but not in strains lacking both the Vi capsule and long O-antigen, suggesting that IgM binds to long O-antigen in the absence of Vi capsule. However, the loss of long O-antigen increases IgM binding to S. Paratyphi A, and this is abrogated in the absence of both long and very long O-antigen. Collectively, these results show that nontyphoidal S. Typhimurium exhibits higher levels of C3b and IgM binding than the enteric fever serovars S. Typhi and S. Paratyphi A, and the determinants of resistance to serum killing, complement deposition and immunoglobulin binding are serovar-dependent. WzzB-dependent long O-antigen production is defective in S . Paratyphi A due to an arginine-98 to cysteine mutation As S. Paratyphi A is entirely dependent on FepE-mediated very long O-antigen for serum resistance, and the loss of WzzB had no measurable impact, we compared O-antigen production among Salmonella serovars by gel electrophoresis ( Fig 2A ). S. Typhimurium and S. Typhi exhibited an expected long O-antigen profile with 16-35 O-antigen repeating units. However, long O-antigen was not visible in extracts from S. Paratyphi A, which instead produces greater quantities of very long O-antigen in comparison to S. Typhimurium. The elimination of FepE-dependent very long O-antigen in S. Paratyphi A increased short O-antigen production but failed to restore long O-antigen production. These findings suggest the WzzB O-antigen chain length regulator is hypofunctional in S. Paratyphi A, resulting in reduced quantities of long O-antigen in S. Paratyphi A and accounting for the lack of an effect of a wzzB null mutation on serum sensitivity in that serovar; this could also account for the dependence of S. Paratyphi A on very long O-antigen for serum resistance ( Fig 1A ). Download figure Open in new tab Fig 2. S. Paratyphi A has deficient long O-antigen production due to R98C mutation in WzzB. (A) LPS extraction followed by gel electrophoresis and silver staining shows that S. Paratyphi A long O-antigen production is deficient, whereas very long O-antigen production is increased compared to S. Typhimurium. (B) Amino acid sequence of WzzB in S. Typhimurium, S. Typhi, and S. Paratyphi A. Mutations shared by S. Typhi and S. Paratyphi A in relation to S. Typhimurium are highlighted in yellow. Mutations unique to S. Paratyphi A WzzB are highlighted in red. (C) Allelic exchange of S. Paratyphi A wzzB with S. Typhimurium or S. Typhi wzzB restores long O-antigen production by S. Paratyphi A. A single amino acid mutation in S. Paratyphi A WzzB from Arg to Cys at position 98 reduces WzzB activity. (D) Restoration of S. Paratyphi A long O-antigen production by functional WzzB confers serum resistance in the absence of very long O-antigen. Complement bactericidal activity was assayed in 25% pooled human serum after 60 min incubation at 37 °C. Statistical analysis was performed by one-way ANOVA on 3 independent replicates, with P values in red indicating statistical significance as P < 0.05. Column bars represent the means, with error bars showing standard deviation. STm, Typhimurium; STy, Typhi; SPa, Paratyphi A; O-ag, O-antigen. Protein sequence comparison showed that the S. Typhi and S. Paratyphi A WzzB proteins differ by three and six non-synonymous point mutations, respectively, compared to S. Typhimurium WzzB ( Fig 2B ). The three mutations in S . Typhi WzzB are shared with S. Paratyphi A, suggesting that these mutations are not responsible for reduced WzzB activity. Allelic exchange was performed to replace the S. Paratyphi A wzzB gene with wzzB from S. Typhimurium or S. Typhi, which restored long O-antigen production in S. Paratyphi A ( Fig 2C ), indicating that one or more of the three unique non-synonymous mutations not found in S. Typhi WzzB is responsible for reduced activity of S. Paratyphi A WzzB. After replacement of each of these residues in S. Typhi WzzB, mutation of Arg98-to-Cys (R98C) was found to reduce the activity of S. Typhi WzzB and produced a long O-antigen profile similar to that of S. Paratyphi A WzzB ( Fig 2C ). The Glu158-to-Lys or Thr177-to-Met mutations did not affect long O-antigen production. The effect of these mutations on S. Paratyphi A serum resistance was also determined ( Fig 2D ). Restoration of S. Paratyphi A long O-antigen production with S . Typhimurium or S . Typhi wzzB allelic exchange increased resistance to human serum when very long O-antigen production was absent. However, the increase in serum resistance was abrogated by a WzzB R98C mutation, in association with a reduction in long O-antigen production. Measurement of resistance to a lower (25%) concentration of human serum showed that the long O-antigen produced by the hypofunctional S. Paratyphi A WzzB protein still provides some resistance to serum killing in comparison to S. Paratyphi A that lacks both WzzB and FepE ( Fig 2D ). Collectively, these data show that the WzzB R98C mutation in S. Paratyphi A reduces long O-antigen production. Restoration of long O-antigen production in S. Paratyphi A reduces very long O-antigen production and affects complement deposition and immunoglobulin binding Long and very long O-antigen vary in chain length but not in composition. The chain length modulators WzzB and FepE compete for the same substrates, but the mechanism of chain length modulation is poorly understood [ 45 ]. Since S. Paratyphi A lacks long O-antigen but exhibits robust very long O-antigen production, we measured O-antigen production by densitometry to determine whether very long O-antigen production is affected by WzzB activity. Restoration of long O-antigen production in S. Paratyphi A by allelic exchange with S. Typhimurium wzzB decreased very long O-antigen production ( Fig 3A and 3B ). Thus, the production of very long O-antigen in S. Paratyphi A appears to be inversely proportional to WzzB activity. Download figure Open in new tab Fig 3. Restoration of S. Paratyphi A long O-antigen reduces very long O-antigen production and impacts IgM binding. (A) Three independent S. Paratyphi A LPS extractions followed by gel electrophoresis and silver staining were performed to show restoration of long O-antigen and reduction of very long O-antigen. (B) Densitometry analysis. Statistical analysis was performed by one sample t test with a hypothetical value of 1.0 from 3 independent LPS extractions, with P values in red indicating statistical significance as P < 0.05. Line in the scatter dot plot represents the mean. (C) LPS extraction followed by gel electrophoresis and silver staining shows that wzzB allelic exchange shifts the modal long O-antigen and restores long O-antigen production in S. Typhimurium and S. Paratyphi A, respectively. (D) wzzB allelic exchange has small but significant effects on C3b deposition in S. Typhimurium and S. Paratyphi A. (E) Restoration of S. Paratyphi A long O-antigen production reduces IgG binding but (F) increases IgM binding. C3b and immunoglobulin binding were determined following 20 min incubation at 37 °C in 50% pooled NHS. Statistical analysis was performed by t test on 3-7 independent replicates, with P values in red indicating statistical significance as P < 0.05. Some of the C3b deposition, IgG and IgM median FI values for STm, STm Δ fepE, SPa, and SPa Δ fepE are from the same experiment shown in Figs. 1B-D but compared here to the corresponding wzzB allelic exchange construct. Column bars represent the means, with error bars showing standard deviation. STm, Typhimurium; STy, Typhi; SPa, Paratyphi A; FI, fluorescence intensity. Since restoration of long O-antigen production by wzzB allelic exchange in S. Paratyphi A decreased very long O-antigen production, we determined whether the restoration of long O-antigen production also affected C3b deposition and immunoglobulin binding. S. Paratyphi A with restored long O-antigen production was found to be more resistant to C3b deposition even though it produces less very long O-antigen ( Fig 3C and 3D ). In contrast, replacement of S. Typhimurium wzzB with hypofunctional wzz B from S. Paratyphi A decreased long O-antigen production and increased C3b deposition. In the absence of FepE-dependent very long O-antigen production, no significant differences in C3b deposition were observed, indicating that the production of long O-antigen is not sufficient to prevent C3b deposition. No differences were observed in IgG binding to S. Typhimurium expressing the hypofunctional WzzB from S. Paratyphi A ( Fig 3E ). However, restoration of long O-antigen production in S. Paratyphi A decreased IgG binding ( Fig 3E ) while increasing IgM binding ( Fig 3F ). Hypofunctional WzzB and paratose-containing O-antigen in S. Paratyphi A reduce IgM binding and complement activation C3b deposition on S. Paratyphi A remains low in comparison to S. Typhimurium and S. Typhi despite a reduction of very long O-antigen in strains with restored WzzB-dependent long O-antigen production ( Fig 3B and 3D ). Therefore, we analyzed additional possible factors contributing to low C3b deposition in this Salmonella serovar. The repeating O-antigen of S. Paratyphi A contains the dideoxyhexose paratose instead of tyvelose, which is present in S. Typhi, due to pseudogenization of rfbE, encoding a CDP-tyvelose epimerase that converts CDP-paratose to CDP-tyvelose. Previous studies have shown that the composition of O-antigen can impact C3b deposition [ 46 – 48 ]. Under our experimental conditions, repairing S. Paratyphi A rfbE by allelic exchange with functional S. Typhi rfbE did not affect the percentage of bacteria with C3b deposition ( Fig 4A ) but lower quantities of C3b were detected on bacteria with C3b deposition ( Fig 4B ). Conversely, restoring long O-antigen production in S. Paratyphi A by allelic exchange with functional wzzB from S. Typhimurium decreased the overall percent of bacteria with C3b deposition ( Fig 4A ) but did not affect the quantity of C3b deposition on these cells ( Fig 4B ). These differences in C3b deposition were not observed in S. Paratyphi A that produced both long tyvelose-containing O-antigen ( Fig 4A and 4B ). Similarly, IgG binding by S. Paratyphi A was reduced when long O-antigen production was restored, with a smaller reduction observed in a strain expressing a functional rfbE gene, but, no significant differences in IgG binding were measured when both wzzB and rfbE were repaired ( Fig 4C ). In contrast, the restoration of either long O-antigen or tyvelose O-antigen production in S. Paratyphi A significantly increased IgM binding ( Fig 4D ), which increased further when both wzzB and rfbE were repaired. The increase in IgM binding as a result of rfbE allelic exchange was not attributable to restored long O-antigen production, suggesting that S. Paratyphi A WzzB remains hypofunctional when the O-antigen contains tyvelose ( Fig 4E ). These changes in complement deposition and immunoglobulin binding did not affect serum resistance in S. Paratyphi A ( Fig 4F ). Collectively, these observations suggest that reduced long O-antigen production and modified O-antigen composition contribute to the reduced IgM binding of S. Paratyphi A. Download figure Open in new tab Fig 4. Reduction of long O-antigen production and antigenic shift to O2-antigen in S. Paratyphi A decreases IgM binding. ( A) and (B) Restored S. Paratyphi A long O-antigen production further reduces complement C3b deposition, but the presence of O9 antigen abrogates this effect. (C) IgG binding is reduced following restoration of long O-antigen or O9-antigen production in S. Paratyphi A. (D) Restoration of long O-antigen and O9-antigen production increases IgM binding of S. Paratyphi A, but (F) does not increase sensitivity to serum killing. Representative flow cytometry histogram in (D) displays increased IgM binding to S. Paratyphi A due to restoration of long O-antigen and O9-antigen. (E) Representative LPS extraction showing that a shift to O9-antigen does not restore long O-antigen production in S. Paratyphi A. C3b and immunoglobulin binding were determined following 20 min incubation at 37 °C in 50% human pooled serum, while serum resistance was determined following 60 min incubation. Statistical analysis was performed by one-way ANOVA on 3-5 independent replicates, with P values in red indicating statistical significance with P <0.05. Column bars represent the means, with error bars showing standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A; FI, fluorescence intensity. Complement activation leads to the production of the soluble anaphylatoxins C3a and C5a. We measured the contribution of O-antigen length and composition on anaphylatoxin production in human serum following exposure to nontyphoidal and enteric fever Salmonella serovars. Differences in complement C3a production among serovars were subtle ( Fig 5A ), but S. Typhimurium induced greater C5a production than the enteric fever serovars ( Fig 5B ). Long O-antigen production in S. Typhimurium was required for increased C5a production, while very long O-antigen was dispensable ( Fig 5B ). Both long O-antigen and the Vi capsule contributed to reduced C3a production following exposure of serum to S. Typhi, but no increase in C5a production was observed in mutant strains. In S. Paratyphi A, very long O-antigen prevented C3a and C5a production, in agreement with our earlier results indicating that very long O-antigen is the primary determinant of complement resistance in S. Paratyphi A. Restoration of long and tyvelose O-antigen production in S. Paratyphi A, which increased IgM binding, also increased C3a and C5a production. These observations suggest that reduced long O-antigen production and modified O-antigen composition contribute to decrease anaphylatoxin elicitation by S. Paratyphi A. Download figure Open in new tab Fig 5. O-antigen length and composition impacts complement activation by Salmonella nontyphoidal and enteric fever serovars. ( A) Restoration of S . Paratyphi A long and O9-antigen increases human C3a and (B) C5a production following incubation with human serum for 60 min at 37°C. The S. Typhi Vi capsule prevents C3a production but does not impact C5a release. Long O-antigen in S. Typhimurium is the primary determinant of C5a release. C3a and C5a release were measured by enzyme-linked immunosorbent assay (ELISA). For statistical comparison, individual replicates are normalized to the corresponding wild-type serovar and statistical analysis performed by one sample t test with a hypothetical value of 1.0, with P values in red indicating statistical significance as P < 0.05. Column bars represent the means, with error bars showing standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A. O-antigen modifications by glycosyltransferases in S. Typhimurium and S. Paratyphi A modulate complement activation and IgM binding O-antigen length and composition are important determinants of IgM binding to S. Paratyphi A ( Fig 4 and Fig 5 ). However, even with increased IgM binding ( Fig 4D ) and complement activation ( Fig 5A and 5B ), S. Paratyphi A exhibits reduced complement deposition compared to S. Typhimurium even though it lacks the S. Typhi Vi capsule. We sought to determine whether S. Typhimurium and S. Paratyphi A O-antigen-modifying glycosyltransferases influence complement activation and deposition, IgM binding, and serum resistance, as these modifications have been previously shown to impact antibody binding and serum resistance in other serovars [ 49 ]. Three O-antigen-modifying glycosyltransferases have been identified in S. Paratyphi A: the OafB acetyltransferase (encoded by SPA0467, also known as family II gtrC or F2gtrC ) acetylates the 2- and 3-hydroxyl groups of rhamnose of the O-antigen repeating unit, and the F1gtrC ( SPA2387 ) and F3gtrC ( SPA2169 ) glycosyltransferases attach a glucosyl group to the 6- or 4-hydroxyl group of galactose in the O-antigen repeating unit, respectively [ 50 – 54 ]. Deletion of OafB and F3gtrC increased C5a release and IgM binding in S. Paratyphi A, but deletion of F1gtrC did not ( Fig 6A and 6B ). Deletion of the acetyltransferase and glycosyltransferases did not impact C3b deposition or serum resistance ( Fig 6C , 6D , and 6E ). Deletion of the abequose acetyltransferase OafA in S. Typhimurium did not impact C5a release but increased IgM binding ( Fig 6F and 6G ) [ 55 , 56 ]. Deletion of the glycosyltransferases F3gtrC or the putative F4gtrC, with unknown O-antigen modifying function, also failed to impact C5a anaphylatoxin release by S. Typhimurium ( Fig 6F ). However, F4gtrC deletion increased IgM binding ( Fig 6G ). Similar to S. Paratyphi A, deletion of these O-antigen modifying enzymes did not impact C3b deposition or serum resistance ( Fig 6H , 6I , and 6J ). Collectively, these results suggest that the effect of O-antigen modifications by acetyltransferases and glycosyltransferases on complement activation and antibody binding are serovar dependent but are not determinants of C3b deposition or serum resistance in S. Paratyphi A and S. Typhimurium. Download figure Open in new tab Fig 6. Effect of O-antigen modifying glycosyltransferases on complement activation and Salmonella interactions with human serum. ( A) O-antigen modifications by glycosyltransferases in S. Paratyphi A decrease complement activation as measured by C5a release and (B) IgM binding. (C) and (D) O-antigen modifications by glycosyltransferases in S. Paratyphi A do not impact complement deposition or (E) serum resistance. (F) O-antigen modifications by glycosyltransferases in S. Typhimurium do not impact complement activation, (H) and (I) C3b deposition, or (J) serum resistance, but (G) reduce IgM binding. Statistical analysis was performed by one-way ANOVA on 3-4 independent replicates, with P values in red indicating statistical significance with P <0.05. Column bars represent the means, with error bars displaying standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A. Multiple gene copies of rfbV in S. Paratyphi A increase very long O-antigen production, prevent C3b deposition, and enhance serum resistance S. Paratyphi A produces greater quantities of very long O-antigen than S. Typhimurium ( Fig 2A ). We hypothesized that increased very long O-antigen production in S. Paratyphi A prevents C3b deposition while reduced very long O-antigen production in S. Typhimurium permits C3b deposition. First, we sought to determine an explanation for increased very long O-antigen levels in S. Paratyphi A. The O-antigen locus of S. Paratyphi A strain ATCC 9150 contains three gene copies of rfbV (also known as wbaV ), which encodes an enzyme that links paratose to mannose of the O-antigen repeating unit before it is flipped to the periplasmic face by RfbX (also known as Wzx); S. Typhimurium and S. Typhi contain only a single copy of rfbV [ 57 – 60 ]. The extra copies of rfbV in S. Paratyphi A are preceded by a hypothetical open reading frame (ORF) composed of the C-terminus of RfbX and the N-terminus of RfbU, which attaches rhamnose to mannose in the O-antigen repeating unit ( Fig 7A ). To determine if increased rfbV gene copy number enhances very long O-antigen to prevent C3b deposition and increase serum resistance, we constructed a S. Paratyphi A strain that contains only one copy of rfbV ( Fig 7A ). The constructed S. Paratyphi A strain with a single copy of rfbV was more susceptible to serum killing compared to the wild-type S. Paratyphi A strain with three copies of rfbV ( Fig 7B ). Serum resistance in the constructed S. Paratyphi A strain with a single copy of rfbV was fully restored by constitutively expressing either rfbV or rfbV preceded by the hypothetical rfbU - rfbX fusion ORF, as found in wild-type S. Paratyphi A ( Fig 7B ). Interestingly, constitutive expression of only the hypothetical rfbU - rfbX fusion ORF also restored some serum resistance, suggesting that the putative fusion protein retains enzymatic activity affecting O-antigen production ( Fig 7B ). Serum killing was proportional to C3b deposition ( Fig 7C ), and IgM binding was increased in the S. Paratyphi A strain carrying a single copy of rfbV ( Fig 7D ). O-antigen staining and densitometry analysis showed that S. Paratyphi A with multiple rfbV copies produced more very long O-antigen than the constructed strain with a single rfbV copy ( Fig 7E and 7F ). Enhanced very long O-antigen production in S. Paratyphi A with a single rfbV copy was restored with constitutive expression of either rfbV or rfbV preceded by the hypothetical rfbU - rfbX fusion ORF, and more modestly increased by expression of only the hypothetical rfbU - rfbX fusion ORF along ( Figure 7E and 7F ). Collectively, these results suggest that rfbV gene duplication in S. Paratyphi A enhances very long O-antigen production to prevent complement deposition, IgM binding, and enhance serum resistance. Download figure Open in new tab Fig 7. rfbV gene triplication in S. Paratyphi A increases very long O-antigen production and reduces C3b deposition. (A) Gene diagram displaying triplicated rfbV gene in wild-type S. Paratyphi A ATCC 9150 and the constructed strain with a single rfbV gene copy. The nonfunctional rfbE gene is displayed by a dashed arrow, and partial genes are denoted by an asterisk. (B) Serum resistance. S. Paratyphi A with a single gene copy of rfbV is less resistant to serum killing compared to wild-type S. Paratyphi A that contains three rfbV gene copies. S. Paratyphi A complement resistance is restored by complementation with rfbV alone or rfbV preceded by the putative fusion protein encoded by incomplete copies of rfbU and rfbX, which are denoted by an asterisk. Complementation with the putative fusion protein composed of Rfb*U*X also confers partial serum resistance. (C) C3b Deposition. S. Paratyphi A with single gene copy of rfbV is more susceptible to C3b deposition. Serum resistance is directly proportional to C3b deposition. (D) IgM Binding. Multiple rfbV gene copies reduce IgM binding. Reduced IgM binding is also observed when the putative Rfb*U*X fusion protein is expressed. (E) Extracted LPS subjected to gel electrophoresis followed by silver staining showing decreased very long O-antigen production in S. Paratyphi A with a single rfbV gene copy compared to wild-type S. Paratyphi A ATCC 9150 with three rfbV gene copies. (F) Densitometry analysis from three independent LPS extractions. Statistical analysis was performed by one sample t test with a hypothetical value of 1.0, with P values in red indicating statistical significance as P < 0.05. Column bars represent the mean with error bars showing standard deviation. (G) LPS extraction followed by silver staining showing increased very long O-antigen production in S. Typhimurium that constitutively expresses fepE . (H) C3b Binding. Increased very long O-antigen production in S. Typhimurium decreases C3b binding, whereas long O-antigen promotes C3b binding. (I) IgM Binding. Increase in very long O-antigen does not affect IgM binding to Typhimurium, whereas long O-antigen increases IgM binding to Typhimurium. Statistical analysis (except for Fig 7F ) was performed by one-way ANOVA on 3-5 independent replicates, with P values in red indicating statistical significance with P <0.05. Column bars represent the means, with error bars showing standard deviation. STm, S. Typhimurium; STy, S. Typhi; SPa, S. Paratyphi A. We reasoned that increasing rfbV production in S. Typhimurium might also increase very long O-antigen production and prevent C3b deposition. However, constitutively expressing rfbV in Typhimurium did not increase very long O-antigen production nor prevent C3b deposition, in contrast to S. Paratyphi A (S1 Fig). A previous study suggested that FepE levels are higher in S. Paratyphi A compared to S. Typhimurium [ 61 ]. To determine whether increased fepE expression can increase very long O antigen production, we constitutively expressed fepE in S. Typhimurium, which resulted in increased very long O-antigen production ( Fig 7G ) and decreased C3b deposition ( Fig 7H ). Long O-antigen length was found to inhibit the anti-opsonic effect of increased very long O-antigen production, since S. Typhimurium with long O-antigen was more susceptible to C3b deposition compared to S. Typhimurium with shorter long O-antigen following allelic exchange with the hypofunctional wzzB from S. Paratyphi A or lacking long O-antigen altogether ( Fig 7H ). Similarly, long O-antigen increased IgM binding ( Fig 7I ). These findings suggest that high levels of very long O-antigen are required for complement evasion, that long O-antigen promotes complement deposition and IgM binding in S. Typhimurium, and that rfbV gene duplication in S. Paratyphi A is required to inhibit complement deposition and IgM binding. Discussion Salmonella enterica lipopolysaccharide, with its repeating O-antigen units, functions as a physicochemical barrier that modulates host recognition, phagocyte ingestion, susceptibility to bactericidal mechanisms, and stimulation of innate immunity via the LPS-binding protein—CD14—TLR4—MD-2 complex [ 28 , 29 , 33 , 62 ]. Important differences in this outermost layer of the cell envelope of nontyphoidal and enteric fever Salmonella serovars result in critical differences in the respective responses to these pathogens that are initiated when they interact with the host. In this study, we have compared the roles of long and very long O-antigen and the Vi capsule in conferring resistance to serum killing, complement and immunoglobulin binding, and complement activation in nontyphoidal S. Typhimurium and the enteric fever serovars S. Typhi and S. Paratyphi A. In agreement with previous studies, we found that long O-antigen confers resistance to serum killing in S. Typhimurium and S. Typhi [ 30 , 31 , 39 , 63 ]. In contrast, WzzB-dependent long O-antigen is not required for serum resistance of S. Paratyphi A when very long O-antigen is present. LPS extractions suggested that S. Paratyphi A has relatively low levels of long O-antigen, and we show that this results from an Arg98-to-Cys mutation that reduces WzzB activity. Hypofunctional WzzB renders S. Paratyphi A reliant on FepE-dependent very long O-antigen for serum resistance, which is in sharp distinction to S. Typhimurium, which does not depend on very long O-antigen for serum resistance, as previously shown [ 29 , 33 , 39 ]. S. Typhi contains a fepE pseudogene and therefore does not produce very long O-antigen, but its acquisition of the SPI-7 encoded Vi capsule provides an analogous structure that confers serum resistance, in agreement with previous observations [ 20 , 64 ]. Thus, nontyphoidal and enteric fever Salmonella serovars have evolved distinct mechanisms to resist serum killing. S. Typhimurium binds the complement component C3b more avidly than S . Typhi or S . Paratyphi A ( Fig 1A ). The S . Typhi Vi capsule has been suggested to resist C3b deposition because it lacks free hydroxyl groups for nucleophilic attack on the susceptible complement C3b thioester bond [ 25 , 32 , 65 ]. O-antigen repeating units containing free hydroxyl groups have been suggested to covalently attach to C3b by this mechanism [ 32 ]. However, the O-antigen repeating units in S . Typhimurium, S . Typhi, and S . Paratyphi A differ only by the stereoisomers abequose, tyvelose, and paratose, respectively, yet S. Paratyphi A, which lacks the Vi capsule, binds less C3b than either S . Typhimurium or S . Typhi ( Fig 1B ). This suggests that additional factors modulate C3b deposition on the Salmonell a surface. The classical pathway of complement activation relies on antigen recognition by immunoglobulins to initiate complement deposition [ 22 ]. Human serum contains IgG and IgM that are able to bind all three Salmonella enterica serovars tested. The loss of O-antigen in S . Typhimurium leads to a decrease in IgG and IgM binding, suggesting that O-antigen contains a major epitope for immunoglobulin recognition. The S . Typhi Vi capsule shields IgG and IgM O-antigen epitope recognition. In S . Paratyphi A, very long O-antigen plays a role analogous to that of the S . Typhi Vi capsule in preventing IgG recognition of long O-antigen. However, IgM epitopes on S . Paratyphi A appear to have a more complex antibody-antigen relationship, in which long O-antigen and the loss of the O9 tyvelose antigen play a critical role. The paratose O2-antigen contributes to the decrease in IgM binding, since repairing the S . Paratyphi A rfbE pseudogene to restore production of the tyvelose O9-antigen significantly increases IgM binding, in agreement with Hiyoshi, et al. [ 20 ]. We have also determined that restoration of S . Paratyphi A long O-antigen production increases IgM binding, which is further increased when combined with restored O9-antigen production. Unexpectedly, increased binding of IgM, which is a potent complement activator, does not enhance complement C3b deposition on S . Paratyphi A but increases anaphylatoxin production. Paradoxically, either deletion of the hypofunctional S. Paratyphi A WzzB or restoration of fully functional WzzB-dependent long O-antigen increased IgM binding. We hypothesize that deletion of the S . Paratyphi A hypofunctional WzzB increases the accessibility of IgM epitopes closer to the bacterial surface, while restoration of long O-antigen provides different IgM epitopes that are more accessible for IgM crosslinking than very long O-antigen. Future studies may examine the physiochemical properties of long and very long O-antigen that modulate immunoglobulin C3b binding to S. Paratyphi A. The present study clarifies and expands upon earlier observations regarding the role of LPS in S. Paratyphi A innate immune evasion. Mylona et al. suggested that increased production of S . Paratyphi A very long O-antigen results from increased fepE expression relative to S . Typhimurium, and that this inhibits inflammasome activation and pyroptotic cell death [ 61 ]. We propose that increased very long O-antigen production is also important for S. Paratyphi A immune evasion because it prevents binding of C3b and immunoglobulin and thereby prevents the recruitment of inflammatory cells by complement-derived anaphylatoxins. We have further shown that the hypofunctional WzzB of S. Paratyphi A is responsible for increased very long O-antigen production, most likely by diverting substrate to FepE. Liu et al. have suggested that the main contributing factor to low levels of long O-antigen in S . Paratyphi A is the inefficient attachment of paratose to the O-antigen repeating unit by the glycosyltransferase RfbV, also known as WbaV [ 66 ]. However, one would expect low levels of both long and very long O-antigen if RfbV were inefficient, but S . Paratyphi A in fact produces robust levels of very long O-antigen. Based on our new findings, we propose that low levels of long O-antigen production in S. Paratyphi A primarily result from hypofunctional WzzB. Multiple rfbV gene copies in S. Paratyphi A are required to produce enhanced levels of very long O-antigen to evade host immune defenses. The importance of multiple rfbV gene copies is supported by recent findings showing that S. Paratyphi A clinical isolates contain more than two rfbV copies, with most preceded by a putative partial rfbU and rfbX gene fusion [ 67 ]. Our results also suggest that the putative RfbU-RfbX fusion protein retains enzymatic activity, since its expression appears sufficient to provide some resistance to serum killing, complement deposition, and decreased IgM binding. We speculate that the putative RfbU-RfbX fusion protein is needed in the presence of multiple rfbV copies in S. Paratyphi A to allow efficient production of the repeating O-antigen unit. Although we were able to restore very long O-antigen production by constitutive expression of the rfbV gene in S. Paratyphi A ( Fig 7E ), most plasmid constructs containing rfbV alone were found to contain mutations within the coding sequence. This suggests that increased RfbV enzymatic activity is not tolerated by S. Paratyphi A unless the putative RfbU-RfbX fusion protein is also present. The S. Paratyphi A constitutive rfbV construct used in this study contained a G nucleotide deletion in the ribosome binding site, which likely lowered RfbV protein to tolerable levels in the absence of the putative RfbU-RfbX fusion protein. In summary, we have systematically compared the roles of long and very long O-antigen and Vi capsule in the interaction of serum complement and antibody with nontyphoidal and enteric fever Salmonella serovars. This first contact between pathogen and host immune effectors has critically important implications for subsequent pathogenic events. All three Salmonella serovars examined in this study resist serum killing but employ different mechanisms to do so. Nontyphoidal S. Typhimurium avidly binds complement and allows complement activation, leading to an inflammatory response that it withstands and exploits. In contrast, the enteric fever serovars S. Typhi and S. Paratyphi employ distinct but functionally analogous mechanisms to resist complement and antibody binding and prevent complement activation, which allows them to evade innate immunity and disseminate via trafficking phagocytes. It has been suggested that S. Typhi lost very long O-antigen production and acquired the Vi capsule in response to the selective pressure imposed by serum components including complement and immunoglobulins [ 18 , 68 , 69 ]. Similarly, selection and convergent evolution have driven S. Paratyphi A to overexpress very long O2-antigen as an analogous barrier to complement, immunoglobulins and phagocytes [ 20 ]. We posit that the same selective pressures favored the Arg98-to-Cys WzzB mutation in S. Paratyphi A to reduce long O-antigen production, which reduces IgM binding and increases very long O-antigen production. S. Paratyphi A very long O-antigen production has been further enhanced by rfbV gene duplication and creation of the partial RfbU-RfbX fusion protein, which reduce complement and IgM binding. Thus, the enteric fever Salmonella serovars have evolved to evade host recognition by convergent pathways, in contrast to the pro-inflammatory nontyphoidal serovar S. Typhimurium. These differences play an important role in the distinctive clinical manifestations of enteric fever and Salmonella enteritis. Materials and Methods Bacterial strains and growth conditions Bacterial strains used in this study are listed in Table S1. Mutant alleles were constructed by λ-Red recombination, as previously described [ 70 – 72 ]. Allelic exchanges were constructed by λ-Red recombination with positive selection for loss of tetracycline resistance, as previously described, with adjusted concentration of fusaric acid to 9 μg mL -1 to select for S. Paratyphi A [ 73 ]. Three non-synonymous single nucleotide polymorphisms (SNPs) in S. Paratyphi A wzzB were separately introduced into S. Typhi wzzB cloned into pBlueScript II SK (+) (pBSIISK, Stratagene), using the QuikChange Lightning kit (Agilent, catalog #210518). The plasmid pBSIISK_ wzz B Typhi with the introduced SNPs was used as template for PCR amplification and allelic exchange of wzzB in S. Paratyphi A. Plasmids and oligonucleotides used in this study to construct bacterial strains are listed in Table S2. Mutant alleles were confirmed by PCR and allelic exchanges confirmed by sequencing. S. enterica cultures were grown in Luria-Bertani Broth (LB; Fisher Scientific) at 37 °C with shaking at 250 rpm. Length of incubation is detailed in subsequent sections. Construction of the S. Paratyphi A strain with a single gene copy of rfbX, rfbV, and rfbU Primers FEGP107 and FEGP108 were used to amplify tetRA from genomic DNA of S. Typhimurium containing a Tn10dTc [del-25] insertion (strain JK18). The amplicon was electroporated into S. Paratyphi A strain ATCC 9150 to replace the rfbX-rfbU genomic region with tetRA by λ-Red homologous recombination. The rfbX - rfbU region from S. Typhi strain Ty2 was amplified using primers FEGP131 and FEGP132 and ligated to 900 bp upstream (amplified using primers FEGP129 and FEGP130) and downstream (amplified using primers FEGP131 and FEGP132) amplicons containing homology arms to the corresponding genomic region in S. Paratyphi A. The amplicons were ligated into BamHI-HF and EcoRI-HF restriction enzyme-digested pFOK using NEBuilder HiFi DNA Assembly (New England Biolabs) and electroporated into donor E. coli strain JKE201, as described [ 74 ]. The donor E. coli strain containing pFOK with rfbX-rfbU from S. Typhi was mated with S. Paratyphi A Δ rfbX-U :: tetRA to promote allelic exchange of the S. Typhi rfbX-rfbU genes into S. Paratyphi A, as described [ 74 ]. A similar strategy was then used to sequentially replace the inserted S. Typhi rfbX, rfbV, and rfbU genes with the corresponding S. Paratyphi A genes. Each inserted S. Typhi gene on the S. Paratyphi A chromosome was replaced by tetRA using λ-Red recombination and then replaced with the S. Paratyphi A homologous gene by conjugation with E. coli JKE201 containing the pFOK plasmid with the replacement gene flanked by homology recombination regions. After a S. Paratyphi A strain with single copies of rfbX, rfbV, and rfbU was constructed, primers FEGP129 and FEGP134 were used to amplify the rfbX-rfbU region containing flanking homology regions, ligated into pFOK, and transformed into E. coli JKE201, before finally conjugating into S. Paratyphi A Δ rfbX-U :: tetRA for allelic exchange of the rfbX-rfbU region in one step. Gene constructs were confirmed by sequencing. To construct constitutively expressed rfbV for complementation in S. Paratyphi A, primers FEGP254 and FEGP255 were used to amplify rfbV from S. Paratyphi A gDNA, and the amplicon was ligated into pJK770 that was digested with NcoI-HF using NEBuilder HiFi DNA Assembly. Similarly, fepE and rfbV from S. Typhimurium, and rfb*U*XV and rfb*U*X from S. Paratyphi A were amplified using primers in Table S2 and ligated into pJK770. Serum bactericidal activity Bacteria were grown in 5 mL LB at 37°C with shaking at 250 rpm for 18 hrs to reach stationary phase. Bacterial optical density at 600 nm (OD 600nm ) was adjusted to 1.0, bacteria were pelleted by centrifugation, supernatant was discarded, and pellets were resuspended in PBS to ∼ 1 x 10 9 CFUs mL -1 . Two-hundred µL of washed bacteria were mixed with 200 μL of human pooled serum (MP Biomedicals, catalog #2930149) in a 1.5 mL Eppendorf tube to attain a 50% serum concentration. For bactericidal experiments in 25% human pooled serum, 200 μL of washed bacteria were mixed with 200 μL of 50% human pooled serum diluted in PBS. Bacteria and serum mixtures were incubated at 37°C for 1 hr. Following incubation, 20 μL of bacteria-serum mixture were added to 180 μL of ice-cold PBS and serial dilutions plated on LB agar. CFU were enumerated following overnight incubation at 37°C. Serum killing was calculated relative to bacteria incubated in parallel with PBS instead of human pooled serum. C3b deposition and immunoglobulin binding Bacteria were incubated in 50% human pooled serum, as described above. After 20 min incubation, 100 μL of bacteria-serum mixture were added to 1 mL ice-cold PBS, pelleted by centrifugation, and supernatant was discarded. Bacteria were incubated with mouse phycoerythrin anti-human C3b/iC3b (BioLegend, catalog #846104), mouse Alexa Fluor ® 488 anti-human IgG Fc (BioLegend, catalog #409322), or mouse Alexa Fluor ® 488 anti-human IgM (BioLegend, catalog #314533) for 30 min on ice. Unbound antibody was washed twice with 1 mL PBS by pelleting bacteria before fixing with 100 μL IC Fixation Buffer (Invitrogen, catalog #00-8222-49) for 20 min at room temperature. Fixed bacteria were washed with 1 mL PBS and resuspended in PBS. Analysis was performed on 50,000 events collected in a BD ® LSR II Flow Cytometer or FACSymphony A3 Cell Analyzer with low sample flow rate. ELISA (Enzyme-linked immunosorbent assay) Bacteria were incubated in 50% human pooled serum as described above. After 60 min incubation at 37°C, bacteria were pelleted by centrifugation at 20,000x g for 3 min at 4°C. The top 200 ul of supernatant were removed and stored at -80°C until analysis. Human C3a and C5a production were measured using the Human Complement C3a ELISA Kit (Invitrogen, catalog #BMS2089) and the Human C5a ELISA Kit (Invitrogen, catalog #BMS2088), respectively, following the manufacturer’s instructions. LPS extraction, staining, and densitometry analysis LPS extraction was performed as previously described [ 75 ]. LPS was separated in a Novex™ WedgeWell™ 14% Tris-Glycine Gel (Invitrogen, catalog#XP00145BOX). Silver staining of extracted LPS was performed as previously described [ 76 ]. Imaged gels were analyzed with Fiji [ 77 ] to obtain area under the curve of the lipid A+core and very long O-antigen bands. Statistical analyses Statistical comparisons were performed using Prism version 9.2.0 (GraphPad). Statistical method and sample size for experiments are detailed in the figure legends. Flow cytometry data were analyzed using FlowJo version 10.7.1 (Beckton Dickinson & Company). Supporting information S1 Fig. Constitutive rfbV expression in S. Typhimurium does not increase very long O-antigen production. (A) LPS extraction followed by gel electrophoresis and silver staining shows that constitutive rfbV expression in S. Typhimurium does not increase very long O-antigen production nor (B) decrease C3b deposition. STm, S. Typhimurium. S2 Fig. Model of serum resistance in Salmonella enterica nontyphoidal serovar Typhimurium and the enteric fever serovars Typhi and Paratyphi A. S. Typhimurium binds serum antibodies and complement inducing a pro-inflammatory response but survives serum bactericidal mechanisms. The enteric fever Salmonella serovars have evolved to evade host recognition by convergent pathways. The S. Typhi Vi capsule shields against complement deposition and antibody binding. S. Paratyphi A, which lacks the Vi capsule, has acquired several mutations that increase the amount of very long O-antigen to protect against complement deposition and antibody binding. 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