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Neisseria gonorrhoeae subverts formin-dependent actin polymerization to colonize human macrophages

['Stanimir S. Ivanov', 'Department Of Microbiology', 'Immunology', 'Louisiana State University Health Sciences Center Shreveport', 'Shreveport', 'Louisiana', 'United States Of America', 'Reneau Castore', 'Department Of Molecular', 'Cellular Physiology']

Date: 2022-02 

Dynamic reorganization of the actin cytoskeleton dictates plasma membrane morphogenesis and is frequently subverted by bacterial pathogens for entry and colonization of host cells. The human-adapted bacterial pathogen Neisseria gonorrhoeae can colonize and replicate when cultured with human macrophages, however the basic understanding of how this process occurs is incomplete. N. gonorrhoeae is the etiological agent of the sexually transmitted disease gonorrhea and tissue resident macrophages are present in the urogenital mucosa, which is colonized by the bacteria. We uncovered that when gonococci colonize macrophages, they can establish an intracellular or a cell surface-associated niche that support bacterial replication independently. Unlike other intracellular bacterial pathogens, which enter host cells as single bacterium, establish an intracellular niche and then replicate, gonococci invade human macrophages as a colony. Individual diplococci are rapidly phagocytosed by macrophages and transported to lysosomes for degradation. However, we found that surface-associated gonococcal colonies of various sizes can invade macrophages by triggering actin skeleton rearrangement resulting in plasma membrane invaginations that slowly engulf the colony. The resulting intracellular membrane-bound organelle supports robust bacterial replication. The gonococci-occupied vacuoles evaded fusion with the endosomal compartment and were enveloped by a network of actin filaments. We demonstrate that gonococcal colonies invade macrophages via a process mechanistically distinct from phagocytosis that is regulated by the actin nucleating factor FMNL3 and is independent of the Arp2/3 complex. Our work provides insights into the gonococci life-cycle in association with human macrophages and defines key host determinants for macrophage colonization.

During infection, the human-adapted bacterial pathogen Neisseria gonorrhoeae and causative agent of gonorrhea can invade the submucosa of the urogenital tract where it encounters tissue-resident innate immune sentinels, such as macrophages and neutrophils. Instead of eliminating gonococci, macrophages support robust bacterial replication. Here, we detail the life cycle of N. gonorrhoeae in association with macrophages and define key regulators that govern the colonization processes. We uncovered that N. gonorrhoeae establishes two distinct subcellular niches that support bacterial replication autonomously–one niche was on the macrophage surface and another one was intracellular. Gonococci subverted the host actin cytoskeleton through the actin nucleating factor FMNL3 to invade colonized macrophages and occupy a membrane-bound intracellular organelle. We propose that N. gonorrhoeae ability to occupy distinct subcellular niches when colonizing macrophages likely confers broad protection against multiple host defense responses.

Funding: This work was supported by: start-up funds from LSU Health Sciences Center-Shreveport to SSI and to AMD; P20GM134974 from National Institute of General Medical Sciences of the National Institutes of Health to AMD; U54 GM104940 from the National Institute of General Medical Sciences of the National Institutes of Health, which funds the Louisiana Clinical and Translational Science Center (LACaTS) to AMD. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Dynamic remodeling of the host actin cytoskeleton by bacterial pathogens is a critical mechanism for host invasion and spreading mediated by both Arp2/3-dependent and formin-dependent mechanisms [ 63 , 64 ]. The Arp2/3 complex initiates branched actin formation by binding to the sides of pre-existing filaments, whereas the formin family of actin nucleators directs the elongation of unbranched actin filaments [ 65 ]. Here, we investigate the molecular processes mediating human macrophage colonization by gonococci and define at the molecular level the distinct subcellular niches occupied by the bacteria that support replication. Also, we provide evidence for a novel mechanism regulated by the host formin FMNL3 that allows bacterial colonies rather than individual diplococci to invade and colonize human macrophages intracellularly.

On host cells as well as inanimate surfaces gonococci aggregate to form colonies as a direct result of dynamic interactions mediated by Opa proteins, gonococcal lipooligosaccharirides, and the type IV pili [ 49 – 51 ]. Gonococci and the closely related meningococci colonize the surface of epithelial cell to form colonies where plasma membrane proximal bacteria are embedded within membrane ruffles enriched in polymerized actin microfilaments, also known as actin plaques [ 52 – 56 ] and are proposed to remain epicellular [ 57 ]. Retraction of type IV pili emanating from surface-associated gonococcal colonies triggers actin polymerization directly underneath the bacterial colony [ 53 , 55 , 56 ]. A number of host cell surface receptors and adhesion molecules (EGFR, CD46, CD44 and ICAM) that engage the cortical actin network also aggregate within the gonococci-induced actin plaques [ 55 , 58 ]. Ezrin–an adaptor protein that link surface receptors with the cortical actin cytoskeleton is also enriched within these structures [ 52 , 55 ]. Actin plaques are likely produced as a result of surface receptor clustering by the gonococci type IV pili as formation depends on the cholesterol content of the plasma membrane [ 59 ]. Internalized diplococci have been observed in epithelial cells; however, they represent a minor fraction of the total cell-associated bacterial population [ 55 ]. On neutrophils, gonococci have been observed on the cell surface as well as intracellularly [ 27 – 29 , 60 – 62 ].

When associated with different host cells, gonococci have been shown to reside within lysosomes [ 12 , 26 ], in the host cytosol [ 12 , 27 , 28 ] and on the cell surface [ 12 , 28 , 29 ]. Gonococci tether to epithelial cells and neutrophils via type IV pili [ 30 – 32 ], which subsequently facilitates the binding of gonococcal adhesins, such as the outer membrane protein PorB [ 33 , 34 ] and the Opacity (Opa) proteins family [ 35 – 38 ], to host surface receptors, such as the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptor family [ 39 ]. Gonococci encode up to 11 opa loci [ 40 , 41 ] which give rise to 7 to 9 unique Opa proteins with distinct binding affinities for the different CEACAM receptors and interactions with CEACAM1, CEACAM3, CEACAM5 and CEACAM6 have been reported [ 42 – 45 ]. The broadly expressed CEACAM1 receptor mediates attachment to neutrophils and epithelial cells [ 45 ], whereas engagement of the neutrophil specific CEACAM3 receptor transports gonococci to a degradative compartment [ 46 – 48 ].

Depending on the exposure route in human infections, gonococci have been found to colonize several mucosa including the genital, ocular, nasopharyngeal and anal [ 6 ]. In tissues, gonococci adhere to the host epithelium, proliferate and invade the submucosal region. Interaction of N.g with epithelial and immune cells, including neutrophils, macrophages, dendritic cells and T cells causes release of inflammatory mediators in animal models and in humans [ 2 , 7 – 9 ]. Gonococci-encoded immune evasion mechanisms, such as high frequency phase and antigenic variation that diversifies cell surface exposed polypeptides [ 10 , 11 ] and facilitate establishment of persistent infections [ 2 ]. Gonococci replication in association with immune phagocytes, including macrophages and neutrophils, as well as resistance to killing has been described [ 12 – 21 ]. Neutrophils recruitment is a central event in gonorrhea progression; however, the role of tissue resident macrophages, comprising ~10% of total leukocytes isolated from the genitourinary mucosa [ 4 ], as well as macrophages recruited to sites of acute gonococcal infections [ 5 ] remain underexplored. Important work using human cervical tissue explants demonstrated that N.g colonizes and invades the ectocervical, transformation zone and endocervical region of the female cervix [ 22 ], which contain high concentrations of macrophages [ 23 ]. Also, macrophages are recruited to the genital tissues [ 24 ] and infiltrate the uterine mucosa [ 25 ] in murine models of gonorrhea. The mechanism of macrophage colonization by gonococci as well as the identity and subcellular localization of the gonococci-occupied niche remain important open questions.

The betaproteobacteria Neisseria gonorrhoeae (N.g) is a highly adapted human colonizer and the etiological agent of the sexually transmitted disease gonorrhea. Recently, gonorrhea has emerged once again as a major global public health problem due to increased incidence as well as rapid emergence of antibiotics resistance [ 1 ]. A high number of gonococcal infections are asymptomatic, particularly in women, and human-to-human transmission maintains the gonococcal reservoir within the human population [ 2 , 3 ]. Gonococci colonization of the urogenital tract often trigger a localized inflammatory response, which in acute symptomatic infections can progress to the production of purulent exudate that contains gonococci, innate immune cells (macrophages and polymorphonuclear leukocytes- PMNs) and exfoliated epithelial cells [ 4 , 5 ]. A disseminated gonococcal infection can lead to pelvic inflammatory disease, scarring of the fallopian tubes, arthritis and endocarditis [ 6 ].

Results

N.g occupies multiple distinct cellular niches on human macrophages The cellular niche in macrophage infections that supports gonococcal replication remains unknown. In previous studies [12], intracellular as well as cell surface-associated gonococci have been observed. Thus, we investigated the subcellular localization of gonococcal colonies because those cellular compartments likely include the niche that supports bacterial replication. In this study, groups of 4–12 bacteria are referred to as a microcolony and groups larger than 13 members are referred to as a colony. A 3D immunofluorescence microscopy analysis of U937 MFs infected with N.g FA1090 for 6 hours was performed, where the cortical actin network was labeled with phalloidin (Fig 3A). Cortical actin can discern intracellular from cell-surface localized bacteria as it is formed in close proximity to the plasma membrane. Based on this criterion, both intracellular and cell surface-associated gonococcal colonies were observed (Figs 3A and S2A–S2D). These distinct colony topologies were confirmed by an alternative inside/out immunofluorescence microscopy approach, in which extracellular bacteria are labeled with two distinct polyclonal anti-N.g antibodies (one used prior to and another used after cell permeabilization), whereas intracellular bacteria are single-labeled because they are accessible only after cell permeabilization (Fig 3B). Interestingly, mosaic colonies consisting of single antibody-stained as well as dual-stained regions within the same colony were frequently observed (Fig 3C). This hybrid colony type was produced by all four gonococcal strains tested (FA1090, MS11, F62, and FA19) in U937 MFs infections (Fig 3D) as well as in infections of human primary monocyte-derived macrophages (hMDMs) (Figs 3E and S2C and S2D). These data demonstrate that gonococcal colonies were not exclusively intra- or extracellular but rather occupied multiple distinct cellular niches on infected human macrophages by a mechanism conserved in primary hMDMs and U937 MFs. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Gonococci colonize distinct cellular compartments. (A-C) U937 MFs infections with N.g FA1090 at MOI = 2. (A) Representative single focal plane of 3D image Z-stacks showing intracellular and cell-surface associated gonococcal colonies at 6 hpi. Side panels show Z-axis reconstruction at the indicated cross-sections. (B-C) Micrographs of colonized macrophages after inside/out staining with anti-N.g antibodies. (B) Single-stained intracellular (red) and double-stained surface-exposed (yellow) gonococcal colonies are shown. (C) Multiple representative partially exposed (hybrid) gonococcal colonies (yellow and red) are shown. (D-E) Quantitative analyses of the relative colony topologies distributions from infections with different N.g strains at 10hpi (D) in U937 MFs and (E) in human primary monocyte-derived macrophages (hMDMs) derived from two distinct donors are shown. (F) Representative z-projections micrographs of N.g colonies formed on artificial surface (poly-L-lysine coated glass coverslips) or in association with U937 MFs that were stained using the inside/out methodology. (G) Analysis of N.g_OUT and N.g_ALL signal colocalization using Pearson’s correlation coefficient (PCC) in each of the three N.g colony types represented in (F). Means ± SD are shown, unpaired T-test analysis. (A-G) Representative experiments from two (E-G) or at least three (A-D) biological replicates are included. (D-E, G) The number of colonies analyzed from each condition is indicated by “n”. https://doi.org/10.1371/journal.ppat.1010184.g003 We considered that the hybrid colony topology could represent a surface-associated colony in which tightly packed outer layer gonococci render innermost bacteria inaccessible to antibody binding. However, this was not the case because inside/out immunostaining of N.g colonies on poly-L-lysin coverslips in the absence of macrophages show that majority of bacteria are accessed equally well by both antibodies (Fig 3F and 3G). We noticed that in colonies with hybrid topology the antibody inaccessible regions penetrate the cortical actin layer and are enveloped by a meshwork of actin filaments (Fig 4A). Moreover, hybrid colonies remain continuous and appear as a single entity even though a clear demarcation between the antibody accessible and inaccessible colony regions is evident by inside/out microscopy (Fig 4B). The most plausible explanation for the hybrid topology is partial internalization, where the extracellular gonococci acts as a plug to seal a plasma membrane invagination harboring the intracellular portion of the colony. We transduced U937 MFs with the genetically encoded fluorescent membrane marker dsRed-mem. Posttranslational palmitoylation of Cys3 and Cys4 targeted dsRed-mem predominantly to the plasma membrane of U937 MFs. Indeed, plasma membrane invaginations co-localize with the actin meshwork at the cell-proximal regions of hybrid gonococcal colonies (Fig 4C). Together, these data indicate that on colonized human macrophages gonococcal colonies can be found: (i) on the cell surface, (ii) within an intracellular compartment and (iii) partially internalized within in a plasma membrane invagination. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Actin microfilaments and membrane invaginations localize at plasma membrane sites colonized by hybrid gonococcal colonies. Micrographs show representative embedded gonococcal colonies on U937 MFs (MOI = 2) at 8hpi (A) and 10hpi (B) after inside/out differential staining. (A) Side panels show Z-axis reconstruction of the indicated cross-section. Inset shows polymerized actin accumulation (observed on 64.6 ± 8% of embedded colonies, n = 54) specifically on the intracellular region of the hybrid colony (arrowhead). (B) Embedded colonies are continuous. A series of single focal planes spanning 2.7μm shows that the transition point—from intracellular to extracellular region of an embedded colony is continuous and bracketed by cortical actin highlighted by the dashed lines. (C) Actin-rich plasma membrane invaginations surround the intracellular region of hybrid colonies (arrowhead). Single focal plane micrograph of a U937 MF stably expressing the plasma membrane-associated fluorescent reporter DsRed-mem and harboring a hybrid gonococcal colony. (A-C) Representative experiments from at least three biological replicates are included. https://doi.org/10.1371/journal.ppat.1010184.g004

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1010184

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