Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii - PubMed (original) (raw)

Identification and characterization of a novel Fusobacterium nucleatum adhesin involved in physical interaction and biofilm formation with Streptococcus gordonii

Bruno P Lima et al. Microbiologyopen. 2017 Jun.

Abstract

To successfully colonize the oral cavity, bacteria must directly or indirectly adhere to available oral surfaces. Fusobacterium nucleatum plays an important role in oral biofilm community development due to its broad adherence abilities, serving as a bridge between members of the oral biofilm that cannot directly bind to each other. In our efforts to characterize the molecular mechanisms utilized by F. nucleatum to physically bind to key members of the oral community, we investigated the involvement of F. nucleatum outer membrane proteins in its ability to bind to the pioneer biofilm colonizer, Streptococcus gordonii. Here, we present evidence that in addition to the previously characterized fusobacterial adhesin RadD, the interaction between F. nucleatum ATCC 23726 and S. gordonii V288 involves a second outer membrane protein, which we named coaggregation mediating protein A (CmpA). We also characterized the role of CmpA in dual-species biofilm formation with S. gordonii V288, evaluated growth-phase-dependent as well as biofilm expression profiles of radD and cmpA, and confirmed an important role for CmpA, especially under biofilm growth conditions. Our findings underscore the complex set of specific interactions involved in physical binding and thus community integration of interacting bacterial species. This complex set of interactions could have critical implications for the formation and maturation of the oral biofilms in vivo, and could provide clues to the mechanism behind the distribution of organisms inside the human oral cavity.

Keywords: Fusobacterium nucleatum; Streptococcus gordonii; Adhesin; interspecies interaction; oral biofilm.

© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1

Quantitative coaggregation of (a) wild‐type (

WT

) Streptococcus gordonii strain V288 and the

WT

Fusobacterium nucleatum strain

ATCC

23726, or the radD mutant derivative, at different phases of growth. (b) Quantitative coaggregation of WT S. gordonii strains (

DL

1, V288,

ATCC

10558, and

ATCC

  1. with

WT

F. nucleatum strain

ATCC

23726, or the radD mutant derivative at exponential growth. Data represent means and standard deviation of percent coaggregation of at least three independent experiments. *p < .05 compared to wild‐type control

Figure 2

Figure 2

Quantitative coaggregation of wild‐type (

WT

) Streptococcus gordonii strain V288 with Fusobacterium nucleatum

ATCC

23726

WT

strain and

OMP

mutant derivatives (radD, Fn254, cmpA, Fn1893, Fn2047, and aim1). Data represent means and standard deviation of percent coaggregation of at least three independent experiments. *p < .05 compared to wild‐type control

Figure 3

Figure 3

(a) Fusobacterium nucleatum radD cmpA double mutant (strain

BL

  1. was constructed by insertion of the inactivation plasmid

pBPL

9 into cmpA (Fn1554) in a radD::catP mutant background. Black arrows indicate the location of primers used for mutant construction and analysis. (b) Confirmation of plasmid insertion into cmpA by

PCR

analysis of the cmpA mutant (m) with wild‐type (WT) as the control

Figure 4

Figure 4

Quantitative coaggregation of wild‐type Streptococcus gordonii strain (a) V288, (b)

ATCC

10558, (c)

DL

1, and (d)

ATCC

51656 with Fusobacterium nucleatum strains: Wild‐type (

WT

) and the mutant derivatives: radD, cmpA, and radD cmpA double mutant. Data represent means and standard deviation of percent coaggregation of at least three independent experiments. *p < .05 compared to wild‐type control

Figure 5

Figure 5

Streptococcus gordonii and Fusobacterium nucleatum dual‐species biofilm: (a) Confocal laser scanning microscopy of syto9‐stained dual‐species biofilm after overnight incubation. S. gordonii (Sg) cells constitutively express mCherry from their chromosome and appear orange/yellow on the images. Wild‐type (

WT

) F. nucleatum (Fn) and its mutant derivatives (radD, cmpA, and radD cmpA double mutant) are strained by syto9‐only and appear green on the images. (b) The presence of the Fn mutant strains in the Sg‐Fn dual‐species biofilm is displayed as the percentage of Fn cells normalized to the number of attached Sg cells/well, compared to that measured with

WT

Fn. Cellular ratios were determined by measuring

DNA

concentration by

qPCR

, targeting the F. nucleatum gene fomA and the S. gordonii gene srtA. At least three independent experiments were performed per strain combination. Each value represents means and standard deviation of at least three independent experiments. *p < .05 compared to wild‐type control

Figure 6

Figure 6

radD and cmpA expression:

WT

Fusobacterium nucleatum was grown in Columbia broth for 25 hr. Cell samples were collected every 3 hr and (a)

OD

600 was measured, as well as (b) radD and cmpA expression by

qRT

‐

PCR

. Gene expression was normalized to rpoB and compare to the first time point. The dashed line was added to aid in the comparison. (c) radD and cmpA expression were also measured in cells from Fn biofilm grown overnight in

SHI

‐

FSMS

. Gene expression was normalized to rpoB and compare to planktonic cells grown under the same conditions. Each value represents means and standard deviation of at least three independent experiments. To aid with visualization, the dashed line represents no change

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