Type IV Pilus Assembly Proficiency and Dynamics Influence Pilin Subunit Phospho-Form Macro- and Microheterogeneity in Neisseria gonorrhoeae

ABSTRACT PilA, the major pilin subunit of Pseudomonas aeruginosa type IV pili (T4P), is a principal structural component. PilA has a conserved C-terminal disulfide-bonded loop (DSL) that has been implicated as the pilus adhesinotope. Structural studies have suggested that DSL is involved in intersubunit interactions within the pilus fiber. PilA mutants with single-residue substitutions, insertions, or deletions in the DSL were tested for pilin stability, pilus assembly, and T4P function. Mutation of either Cys residue of the DSL resulted in pilins that were unable to assemble into fibers. Ala replacements of the intervening residues had a range of effects on assembly or function, as measured by changes in surface pilus expression and twitching motility. Modification of the C-terminal P-X-X-C type II beta-turn motif, which is one of the few highly conserved features in pilins across various species, caused profound defects in assembly and twitching motility. Expression of pilins with suspected assembly defects in a pilA pilT double mutant unable to retract T4P allowed us to verify which subunits were physically unable to assemble. Use of two different PilA antibodies showed that the DSL may be an immunodominant epitope in intact pili compared with pilin monomers. Sequence diversity of the type IVa pilins likely reflects an evolutionary compromise between retention of function and antigenic variation. The consequences of DSL sequence changes should be evaluated in the intact protein since it is technically feasible to generate DSL-mimetic peptides with mutations that will not appear in the natural repertoire due to their deleterious effects on assembly.

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An inhibitor of DNA binding and uptake events dictates the proficiency of genetic transformation in Neisseria gonorrhoeae: mechanism of action and links to Type IV pilus expression

Molecular Microbiology ◽

10.1046/j.1365-2958.2002.03265.x ◽

2002 ◽

Vol 46 (5) ◽

pp. 1441-1450 ◽

Cited By ~ 50

Author(s):

Finn Erik Aas ◽

Cecilia Løvold ◽

Michael Koomey

Keyword(s):

Dna Binding ◽

Neisseria Gonorrhoeae ◽

Genetic Transformation ◽

Mechanism Of Action ◽

Type Iv Pilus ◽

Type Iv ◽

Inhibitor Of Dna Binding

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Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly

Molecular Microbiology ◽

10.1111/j.1365-2958.2012.08062.x ◽

2012 ◽

Vol 84 (5) ◽

pp. 857-873 ◽

Cited By ~ 59

Author(s):

Michaella Georgiadou ◽

Marta Castagnini ◽

Gouzel Karimova ◽

Daniel Ladant ◽

Vladimir Pelicic

Keyword(s):

Neisseria Meningitidis ◽

Large Scale ◽

Type Iv Pilus ◽

Type Iv ◽

Large Scale Study ◽

Pilus Assembly

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Functional analyses of pilin-like proteins from Francisella tularensis: complementation of type IV pilus phenotypes in Neisseria gonorrhoeae

Microbiology ◽

10.1099/mic.0.028183-0 ◽

2009 ◽

Vol 155 (8) ◽

pp. 2546-2559 ◽

Cited By ~ 16

Author(s):

Emelie Salomonsson ◽

Åke Forsberg ◽

Norbert Roos ◽

Claudia Holz ◽

Berenike Maier ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Francisella Tularensis ◽

Molecular Mechanisms ◽

Indirect Evidence ◽

Type Iv ◽

Pilin Subunit ◽

Surface Localization ◽

Protein Secretion System ◽

And Function ◽

Functional Analyses

Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for Francisella pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure–function relationships. In this study we expressed a number of Francisella pilin genes in the Tfp-expressing pathogen Neisseria gonorrhoeae lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from Francisella tularensis subspecies tularensis and novicida, were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of Francisella PilA pilins also partially restored competence for natural transformation in N. gonorrhoeae. This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for Francisella PilA proteins to express functional Tfp.

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Discovery of a new Neisseria gonorrhoeae Type IV pilus assembly factor, TfpC

10.1101/2020.09.26.314724 ◽

2020 ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Sexually Transmitted Infection ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Sexually Transmitted ◽

Type Iv

AbstractNeisseria gonorrhoeae rely on Type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection, gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the Type IV pilus in its extended, non-retracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show there are orthologues in numerous bacteria species but not all Type IV pilin expressing bacteria contain orthologous genes. Co-evolution and NMR analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region and a highly charge C-terminal coiled-coil domain.ImportanceMost bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae Type IV pilus is a major virulence and colonization factor for the sexually transmitted infection, gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain the Type IV pili on the bacterial cell surface. There are similar proteins found in the other members of the Neisseria genus and many other bacterial species important for human health.

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Discovery of a New Neisseria gonorrhoeae Type IV Pilus Assembly Factor, TfpC

mBio ◽

10.1128/mbio.02528-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Linda I. Hu ◽

Shaohui Yin ◽

Egon A. Ozer ◽

Lee Sewell ◽

Saima Rehman ◽

...

Keyword(s):

Cell Surface ◽

Neisseria Gonorrhoeae ◽

Bacterial Cell ◽

Bacterial Species ◽

Type Iv Pili ◽

Type Iv Pilus ◽

Transmitted Infection ◽

Content Type ◽

Sexually Transmitted ◽

Type Iv

ABSTRACT Neisseria gonorrhoeae relies on type IV pili (T4p) to promote colonization of their human host and to cause the sexually transmitted infection gonorrhea. This organelle cycles through a process of extension and retraction back into the bacterial cell. Through a genetic screen, we identified the NGO0783 locus of N. gonorrhoeae strain FA1090 as containing a gene encoding a protein required to stabilize the type IV pilus in its extended, nonretracted conformation. We have named the gene tfpC and the protein TfpC. Deletion of tfpC produces a nonpiliated colony morphology, and immuno-transmission electron microscopy confirms that the pili are lost in the ΔtfpC mutant, although there is some pilin detected near the bacterial cell surface. A copy of the tfpC gene expressed from a lac promoter restores pilus expression and related phenotypes. A ΔtfpC mutant shows reduced levels of pilin protein, but complementation with a tfpC gene restored pilin to normal levels. Bioinformatic searches show that there are orthologues in numerous bacterial species, but not all type IV pilin-expressing bacteria contain orthologous genes. Coevolution and nuclear magnetic resonance (NMR) analysis indicates that TfpC contains an N-terminal transmembrane helix, a substantial extended/unstructured region, and a highly charged C-terminal coiled-coil domain. IMPORTANCE Most bacterial species express one or more extracellular organelles called pili/fimbriae that are required for many properties of each bacterial cell. The Neisseria gonorrhoeae type IV pilus is a major virulence and colonization factor for the sexually transmitted infection gonorrhea. We have discovered a new protein of Neisseria gonorrhoeae called TfpC that is required to maintain type IV pili on the bacterial cell surface. There are similar proteins found in other members of the Neisseria genus and many other bacterial species important for human health.

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Attenuation of the Type IV Pilus Retraction Motor Influences Neisseria gonorrhoeae Social and Infection Behavior

mBio ◽

10.1128/mbio.01994-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 10

Author(s):

Alyson M. Hockenberry ◽

Danielle M. Hutchens ◽

Al Agellon ◽

Magdalene So

Keyword(s):

Epidermal Growth Factor Receptor ◽

Growth Factor ◽

Social Behavior ◽

Epidermal Growth Factor ◽

Neisseria Gonorrhoeae ◽

Enzymatic Activity ◽

Growth Factor Receptor ◽

Type Iv Pilus ◽

Type Iv ◽

Epidermal Growth

ABSTRACT Retraction of the type IV pilus (Tfp) mediates DNA uptake, motility, and social and infection behavior in a wide variety of prokaryotes. To date, investigations into Tfp retraction-dependent activities have used a mutant deleted of PilT, the ATPase motor protein that causes the pilus fiber to retract. Δ pilT cells are nontransformable, nonmotile, and cannot aggregate into microcolonies. We tested the hypothesis that these retraction-dependent activities are sensitive to the strength of PilT enzymatic activity by using the pathogen Neisseria gonorrhoeae as a model. We constructed an N. gonorrhoeae mutant with an amino acid substitution in the PilT Walker B box (a substitution of cysteine for leucine at position 201, encoded by pilT L201C ). Purified PilT L201C forms a native hexamer, but mutant hexamers hydrolyze ATP at half the maximal rate. N. gonorrhoeae pilT L201C cells produce Tfp fibers, crawl at the same speed as the wild-type (wt) parent, and are equally transformable. However, the social behavior of pilT L201C cells is intermediate between the behaviors of wt and Δ pilT cells. The infection behavior of pilT L201C is also defective, due to its failure to activate the epidermal growth factor receptor (EGFR)-heparin-binding EGF-like growth factor (HB-EGF) pathway. Our study indicates that pilus retraction, per se , is not sufficient for N. gonorrhoeae microcolony formation or infectivity; rather, these activities are sensitive to the strength of PilT enzymatic activity. We discuss the implications of these findings for Neisseria pathogenesis in the context of mechanobiology. IMPORTANCE Type IV pili are fibers expressed on the surface of many bacteria. Neisseria gonorrhoeae cells crawl, take up DNA, and communicate with each other and with human cells by retracting these fibers. Here, we show that an N. gonorrhoeae mutant expressing an enzymatically weakened type IV pilus retraction motor still crawls and takes up DNA normally. However, mutant cells exhibit abnormal social behavior, and they are less infective because they fail to activate the epidermal growth factor receptor. Our study shows that N. gonorrhoeae social and infection behaviors are sensitive to the strength of the retraction motor enzyme.

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