Polymerization of C9 enhances bacterial cell envelope damage and killing by membrane attack complex pores

Complement proteins can form membrane attack complex (MAC) pores that directly kill Gram-negative bacteria. MAC pores assemble by stepwise binding of C5b, C6, C7, C8 and finally C9, which can polymerize into a transmembrane ring of up to 18 C9 monomers. It is still unclear if the assembly of a polymeric-C9 ring is necessary to sufficiently damage the bacterial cell envelope to kill bacteria. In this paper, polymerization of C9 was prevented without affecting binding of C9 to C5b-8, by locking the first transmembrane helix domain of C9. Using this system, we show that polymerization of C9 strongly enhanced damage to both the bacterial outer and inner membrane, resulting in more rapid killing of several Escherichia coli and Klebsiella strains in serum. By comparing binding of wildtype and ‘locked’ C9 by flow cytometry, we also show that polymerization of C9 is impaired when the amount of available C9 per C5b-8 is limited. This suggests that an excess of C9 is required to efficiently form polymeric-C9. Finally, we show that polymerization of C9 was impaired on complement-resistant E. coli strains that survive killing by MAC pores. This suggests that these bacteria can specifically block polymerization of C9. All tested complement-resistant E. coli expressed LPS O-antigen (O-Ag), compared to only one out of four complement-sensitive E. coli. By restoring O-Ag expression in an O-Ag negative strain, we show that the O-Ag impairs polymerization of C9 and results in complement-resistance. Altogether, these insights are important to understand how MAC pores kill bacteria and how bacterial pathogens can resist MAC-dependent killing.

The Invasin and Complement-Resistance Protein Rck of Salmonella is More Widely Distributed than Previously Expected

The foodborne pathogen Salmonella is responsible for a wide variety of pathologies depending on the infected host, the infecting serovars, and its set of virulence factors. However, the implication of each of these virulence factors and their role in the specific host-pathogen interplay are not fully understood.

Factor H binding protein (fHbp)-mediated differential complement resistance of a serogroup C Neisseria meningitidis isolate from cerebrospinal fluid of a patient with invasive meningococcal disease

During an outbreak of invasive meningococcal disease (IMD) at the University of Southampton, UK, in 1997, two Neisseria meningitidis serogroup C isolates were retrieved from a student (‘Case’), who died of IMD, and a close contact (‘Carrier’) who, after mouth-to-mouth resuscitation on the deceased, did not contract the disease. Genomic comparison of the isolates demonstrated extensive nucleotide sequence identity, with differences identified in eight genes. Here, comparative proteomics was used to measure differential protein expression between the isolates and investigate whether the differences contributed to the clinical outcomes. A total of six proteins were differentially expressed: four proteins (methylcitrate synthase, PrpC; hypothetical integral membrane protein, Imp; fructose-1,6-bisphosphate aldolase, Fba; aldehyde dehydrogenase A, AldA) were upregulated in the Case isolate, while one protein (Type IV pilus-associated protein, PilC2) was downregulated. Peptides for factor H binding protein (fHbp), a major virulence factor and antigenic protein, were only detected in the Case, with a single base deletion (ΔT366) in the Carrier fHbp causing lack of its expression. Expression of fHbp resulted in an increased resistance of the Case isolate to complement-mediated killing in serum. Complementation of fHbp expression in the Carrier increased its serum resistance by approximately 8-fold. Moreover, a higher serum bactericidal antibody titre was seen for the Case isolate when using sera from mice immunized with Bexsero (GlaxoSmithKline), a vaccine containing fHbp as an antigenic component. This study highlights the role of fHbp in the differential complement resistance of the Case and the Carrier isolates. Expression of fHbp in the Case resulted in its increased survival in serum, possibly leading to active proliferation of the bacteria in blood and death of the student through IMD. Moreover, enhanced killing of the Case isolate by sera raised against an fHbp-containing vaccine, Bexsero, underlines the role and importance of fHbp in infection and immunity.

(p)ppGpp-dependent regulation of the nucleotide hydrolase PpnN confers complement resistance in Salmonella enterica serovar Typhimurium

The stringent response is an essential mechanism of metabolic reprogramming during environmental stress that is mediated by the nucleotide alarmones, guanosine tetraphosphate and pentaphosphate ((p)ppGpp). In addition to physiological adaptations, (p)ppGpp also regulates virulence programs in pathogenic bacteria including Salmonella enterica serovar Typhimurium. S. Typhimurium is a common cause of acute gastroenteritis, but it may also spread to systemic tissues resulting in severe clinical outcomes. During infection, S. Typhimurium encounters a broad repertoire of immune defenses that it must evade for successful host infection. Here, we examined the role of the stringent response in S. Typhimurium resistance to complement-mediated killing and found that the (p)ppGpp synthetase-hydrolase, SpoT, is required for bacterial survival in human serum. We identified the nucleotide hydrolase, PpnN, as a target of the stringent response that is required to promote bacterial fitness in serum. Using chromatography and mass spectrometry, we show that PpnN hydrolyzes purine and pyrimidine monophosphates to generate free nucleobases and ribose 5′-phosphate, and that this metabolic activity is required for conferring resistance to complement killing. In addition to PpnN, we show that (p)ppGpp is required for the biosynthesis of the very long and long O-antigen in the outer membrane known to be important for complement resistance. Our results provide new insights into the role of the stringent response in mediating evasion of the innate immune system by pathogenic bacteria.

Genomic Profiling Reveals Distinct Routes To Complement Resistance in Klebsiella pneumoniae

ABSTRACT The serum complement system is a first line of defense against bacterial invaders. Resistance to killing by serum enhances the capacity of Klebsiella pneumoniae to cause infection, but it is an incompletely understood virulence trait. Identifying and characterizing the factors responsible for preventing activation of, and killing by, serum complement could inform new approaches to treatment of K. pneumoniae infections. Here, we used functional genomic profiling to define the genetic basis of complement resistance in four diverse serum-resistant K. pneumoniae strains (NTUH-K2044, B5055, ATCC 43816, and RH201207), and explored their recognition by key complement components. More than 90 genes contributed to resistance in one or more strains, but only three, rfaH, lpp, and arnD, were common to all four strains. Deletion of the antiterminator rfaH, which controls the expression of capsule and O side chains, resulted in dramatic complement resistance reductions in all strains. The murein lipoprotein gene lpp promoted capsule retention through a mechanism dependent on its C-terminal lysine residue; its deletion led to modest reductions in complement resistance. Binding experiments with the complement components C3b and C5b-9 showed that the underlying mechanism of evasion varied in the four strains: B5055 and NTUH-K2044 appeared to bypass recognition by complement entirely, while ATCC 43816 and RH201207 were able to resist killing despite being associated with substantial levels of C5b-9. All rfaH and lpp mutants bound C3b and C5b-9 in large quantities. Our findings show that, even among this small selection of isolates, K. pneumoniae adopts differing mechanisms and utilizes distinct gene sets to avoid complement attack.